Recombinant nucleic acid molecule and application thereof in preparation of circular rna

ABSTRACT

The present disclosure relates to a recombinant nucleic acid molecule and an application thereof in preparation of a circular RNA, and in particular, to a recombinant nucleic acid molecule for preparing a circular RNA, a recombinant expression vector, a circular RNA, a composition, a method for preparing a circular RNA, a method for expressing a target polypeptide in a cell, a method for screening a target coding region sequence, a system for screening a target coding region sequence, and a method for screening a ribozyme recognition site sequence. The recombinant nucleic acid molecule provided by the present disclosure provides a Clean PIE system with a novel structure for preparing a circRNA in vitro, which can avoid introducing additional exon sequences into the circular RNA, improve the sequence accuracy of circular RNA molecules, reduce changes in a secondary structure of the circular RNA, and further reduce the immunogenicity of the circular RNA, and has a good application prospects in the fields of nucleic acid vaccines, expression of therapeutic proteins, gene therapy, and the like.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is based upon and claims the benefit of a priority of Chinese Patent Application No. 202210200112.6, filed on Mar. 2, 2022, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to the fields of molecular biology and bioengineering, and in particular, to a recombinant nucleic acid molecule for preparing a circular RNA, a recombinant expression vector, a circular RNA, a composition, a method for preparing a circular RNA, a method for expressing a target polypeptide in a cell, a method for preventing or treating diseases, a method for screening a target coding region sequence, a system for screening a target coding region sequence, and a method for screening a ribozyme recognition site sequence.

BACKGROUND

A clinical experiment by the National Institutes of Health (NIH) observed that symptoms of adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID) were significantly improved by delivering normal ADA enzyme into children with ADA-SCID ^([1-2]), which has greatly facilitated the clinical research development of gene therapy. With the emergence of gene therapy technology, it is expected to fundamentally cure some diseases that cannot be solved by the existing traditional therapies, and make up for the deficiencies of traditional therapies. However, in the early study of gene therapy, retroviruses were typically used as delivery vectors of target genes to integrate a target fragment in the genome of a target cell through random insertion, presenting great uncertainty and danger. In recent years, with the improvement of viral vectors (lentiviral vectors, adenoviral vectors, recombinant adeno-associated virus vectors, and the like) and the development of non-viral vectors (liposome technology, lipid nanoparticles technology, microsphere technology, dendrimer technology, exosomes, and the like), gene therapy has gained interest again.

Messenger ribonucleic acid (mRNA) is transcribed from DNA and provides necessary genetic information for the next step of protein translation, with important application value in protein production, gene therapy approaches such as serving as nucleic acid vaccines, and the like. As compared with traditional vaccines, nucleic acid vaccines have various advantages such as long-lasting immune response, simple manufacturing process, and applications in tumor prevention, thus showing broad prospects in the fields of preventing acute infectious diseases, HIV, cancer, and others. Particularly, since the outbreak of the novel corona virus disease 2019 (COVID-19), research and development on nucleic acid vaccines has been significantly accelerated.

Circular RNAs (circRNAs) have been proved to synthesize protein mediated by internal ribosome entry sites (IRES) in a cap-independent manner in vitro ^([15]). However, most researchers still have long believed that ribosomes of eukaryotes are unable to translate circRNAs in vivo. With the emergence of RNA sequencing technology (RNA-seq), many more circRNAs have been identified ^([3-8]), leading to increasingly more attention on their study. In addition, with the deepening of research, researchers have found that in eukaryotes, circRNAs are not only widely abundant, but also highly conserved ^([9]). The 5′ and 3′ ends in a circRNA are ligated to each other to form a closed ring, which allows circRNA to show a higher resistance to RNase and thus to enable a longer-acting and lasting expression compared to a linear mRNA ^([10]). In addition, circRNAs may be prepared more efficiently, rapidly, and cost effectively compared to the preparation of linear mRNAs featuring fussy capping, tailing and nucleotide modification. Because of these characteristics, although circRNA is a bran-new gene therapy method, it has been used as a vector for gene therapy for business development.

RNA circularization is a key step in preparation of circRNA. In prior art, in vivo circularization and in vitro circularization are two common methods for circularization. In eukaryotes, a spliceosome splices introns out of an immature mRNA by a method including two steps: attacking, by a 2′-hydroxy group on a specific adenosine (branch point Adenosine, bpA) in an intron, the 5′ splice site to form a 3′-hydroxy group at an exon at the 5′ end; and then, further attacking, by the newly formed 3′-hydroxy end, a 3′ splice site with the assistance of a spliceosome, to form a linear RNA with two exons ligated together and a lariat structure. In this process, a natural circRNA is produced by back-splicing or exon skipping ^([3,10-11]) Although in vivo circularization may ensure the sequence accuracy of the circularized circRNAs, it is still necessary to introduce plasmids into the body as therapeutics, which greatly increases the risk of integration into the genome.

In vitro circularization of RNA mainly depends on formation of a phosphodiester bond, and the most common in vitro RNA circularization methods include mainly a chemical method and an enzymatic method. In case of the chemical circularization method, a natural phosphodiester bond is mainly formed and circularized through a condensation reaction between 5′-monophosphate and 3′-hydroxyl of RNA, catalyzed by cyanogen bromide or ethyl-3-(3′-dimethylaminopropyl)-carbodiimide. However, the chemical circularization method is less efficient in ligating, and is only suitable for ligating small fragments of circRNAs ^([12]). In addition, the chemical groups have great potential safety hazards in gene therapy as well. Therefore, it is difficult for the chemical circularization method to be widely used.

The enzymatic method may be further divided into protease catalyzation and ribozyme catalyzation, where the protease catalyzation mainly includes catalyzing formation of the phosphodiester bond through a splint chain by T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, and RtcB ^([13]). However, the current protease catalyzation ligation methods are less efficient in ligating large fragments of circRNAs, and it is also difficult to obtain circular messenger ribonucleotides with accurate nucleic acid sequences.

Ribozymes are a class of RNAs capable of acting as catalysts similar to proteases. There are usually three methods for preparing circRNAs by ribozyme in vitro: self-splicing of Group I intron, self-splicing of group II intron, and circularization by some subviral genomes. In case of the group II intron, a circle is typically ligated and formed via a 2′-5′diphosphate bond, whose impacts on the expression of circRNAs still needs further study. In case of ligating a circRNA through the genome of a subvirus, ribozymes in the genome of the subvirus are typically required to be introduced. As a result, some RNAs in vivo may usually become potential targets of splicing by these ribozymes. In the industry, it is a common circularization strategy to circularize circular ribonucleotides through the catalysis of the Group I introns, where Anabaena PIE (prematured intron exon) and T4td (thymidylate synthase of T4) PIE are the most widely used ribozyme-catalyzed self-splicing circularization systems. In the presence of guanine and divalent cations, the intron sequences of Anabaena PIE and T4td PIE form a specific structure to be spliced by self-catalyzation, thus allowing the ribonucleotide sequence in the middle of the intron to form a circle.

In the prior art, circRNAs show important application prospects in the fields of gene therapy vectors, in vivo expression of therapeutic proteins, and nucleic acid vaccines, but many unsolved or undiscovered problems still exist. The accuracy of the circRNA sequence is the key to its clinical application, and an important guarantee for the efficacy and safety of subsequent therapy. It has been studied of the effects of ligating and forming circRNAs with the T4 ligase, T4 td PIE system, and Anabaena PIE system on the RNA secondary structure. As shown in FIG. 1 , compared with circularization with T4 ligase, circularization through the self-splicing of the T4 td PIE system and the Anabaena PIE system may result in introduction of additional exon sequences (E1, E2) into the circRNAs, which may result in substantial change in structural conformation of the circularized RNAs. Furthermore, due to the introduction of additional E1 and E2 sequences, the circRNAs obtained by using the T4 td PIE and the Anabaena PIE may trigger immune response in cells, resulting in degradation of the circRNA molecules in cells ^([14]).

Chinese Patent No. CN 112399860 A discloses a vector for making circRNA, wherein the vector comprises the following elements operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ Group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or non-coding region, e.) optionally, a 3′ spacer sequence, f.) a 5′ Group I intron fragments containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. The vector allows production of a circRNA that is translatable or biologically active inside eukaryotic cells. Although the vector may be used to prepare a circular RNA through self-splicing characteristic of PIE system, it is necessary to insert specific exon sequences into the vector to guide the splicing of intron fragments. In addition, introduction of additional exon sequences into the finally obtained circular RNAs may reduce the sequence accuracy of the circular RNAs, resulting in substantial changes in the structural conformation of the circularized RNAs, triggering immune response in cells, inducing degradation of circular RNA molecules in cells, posing potential safety hazards for circular RNAs in nucleic acid vaccines and gene therapy, thereby limiting application of circular RNAs in clinical disease treatment.

REFERENCES

-   [1] Kantoff P W, Kohn D B₁₂, Mitsuya H, et al. Correction of     adenosine deaminase deficiency in cultured human T and B cells by     retrovirus-mediated gene transfer [J]. Proceedings of the National     Academy of Sciences, 1986, 83(17): 6563-6567. -   [2] Kohn D B, Mitsuya H, Ballow M, et al. Establishment and     characterization of adenosine deaminase-deficient human T cell lines     [J]. The Journal of Immunology, 1989, 142(11): 3971-3977. -   [3] Kelly S, Greenman C, Cook P R, et al. Exon skipping is     correlated with exon circularization [J]. Journal of molecular     biology, 2015, 427(15): 2414-2417. -   [4] Djebali S, Davis C A, Merkel A, et al. Landscape of     transcription in human cells [J]. Nature, 2012, 489(7414): 101-108. -   [5] Guttman M, Garber M, Levin J Z, et al. Ab initio reconstruction     of cell type— specific transcriptomes in mouse reveals the conserved     multi-exonic structure of lincRNAs [J]. Nature biotechnology, 2010,     28(5): 503-510. -   [6] Mortazavi A, Williams B A, McCue K, et al. Mapping and     quantifying mammalian transcriptomes by RNA-Seq[J]. Nature methods,     2008, 5(7): 621-628. -   [7] Wang E T, Sandberg R, Luo S, et al. Alternative isoform     regulation in human tissue transcriptomes[J]. Nature, 2008,     456(7221): 470-476. -   [8] Wilusz J E, Sunwoo H, Spector D L. Long noncoding RNAs:     functional surprises from the RNA world [J]. Genes & development,     2009, 23(13): 1494-1504. -   [9] Wilusz J E. A 360 view of circular RNAs: from biogenesis to     functions [J]. Wiley Interdisciplinary Reviews: RNA, 2018, 9(4):     e1478. -   [10] Jeck W R, Sharpless N E. Detecting and characterizing circular     RNAs [J]. Nature biotechnology, 2014, 32(5): 453-461. -   [11] Wang Y, Wang Z. Efficient backsplicing produces translatable     circular mRNAs [J]. RNA, 2015, 21(2): 172-179. -   [12] Gaglione M, Di Fabio G, Messere A. Current Methods in Synthesis     of Cyclic Oligonucleotides and Analogues [J]. Current Organic     Chemistry, 2012, 16(11): 1371-1389. -   [13] Obi P, Chen Y G. The Design and Synthesis of Circular RNAs [J].     Methods, 2021. -   [14] Liu C X, Guo S K, Nan F, et al. RNA circles with minimized     immunogenicity as potent PKR inhibitors [J]. Molecular Cell, 2021. -   [15] Chen C, Sarnow P. Initiation of protein synthesis by the     eukaryotic translational apparatus on circular RNAs [J]. Science,     1995, 268(5209): 415-417.

SEQUENCE LISTING

This application contains a sequence listing that has been submitted electronically as an XML file named “53596-0005001.XML.” The XML file, created on Jul. 19, 2022, s 145,622 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

SUMMARY Problems to be Solved by the Disclosure

In view of the problems existing in the prior art, such as inclusion of additional exon sequences in circular RNAs caused by using PIE system to prepare circular RNAs in vitro in the prior art method, resulting in changes in structural conformation of the circular RNAs, which leads to cellular immune response and reduction of the circular RNA molecules in vivo, posing potential safety hazards in nucleic acid vaccines and gene therapy, the present disclosure provides a recombinant nucleic acid molecule for preparing a circular RNA in vitro.

The circular RNA prepared using it can avoid introduction of additional exon sequences, improve sequence accuracy of circular RNA molecules, reduce changes in secondary structure of circular RNAs, and further reduce immunogenicity of circular RNAs, improve stability of circular RNAs in cells, and reduce safety risks of circular RNAs in clinical application, thus showing wide application prospects in fields of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell (DC) tumor vaccines, mRNA-based gene therapy, protein supplement therapy, and the like.

Means for Solving the Problems

(1) A recombinant nucleic acid molecule for preparing a circular RNA, the recombinant nucleic acid molecule being selected from any one of (i) to (ii):

-   -   (i) the recombinant nucleic acid molecule including elements         arranged in the following order in the 5′ to 3′ direction:     -   an intron fragment II, a truncated fragment II of a coding         element, a translation initiation element, a truncated fragment         I of a coding element, and an intron fragment I;     -   where, a 3′ end of the truncated fragment I of the coding         element includes a ribozyme recognition site I that consists of         a first predetermined number of nucleotides located at the 3′         end of the truncated fragment I of the coding element;     -   a 5′ end of the truncated fragment II of the coding element         includes a ribozyme recognition site II that consists of a         second predetermined number of nucleotides located at the 5′ end         of the truncated fragment II of the coding element;     -   the nucleotide sequence of the truncated fragment I of the         coding element and the nucleotide sequence of the truncated         fragment II of the coding element form a coding element sequence         encoding at least one target polypeptide in the 5′ to 3′         direction; the nucleotide sequence of the truncated fragment I         of the coding element corresponds to a partial sequence close to         the 5′ direction in the coding element sequence, and the         nucleotide sequence of the truncated fragment II of the coding         element corresponds to a remaining partial sequence close to the         3′ direction in the coding element sequence;     -   the nucleotide sequence of the intron fragment I and the         nucleotide sequence of the intron fragment II form an intron         sequence in the 5′ to 3′ direction; the nucleotide sequence of         the intron fragment I includes a partial sequence close to the         5′ direction in the intron sequence, and the nucleotide sequence         of the intron fragment II includes a remaining partial sequence         close to the 3′ direction in the intron sequence;     -   alternatively, (ii) the recombinant nucleic acid molecule         including elements arranged in the following order in the 5′ to         3′ direction:     -   an intron fragment III, a truncated fragment IV of a coding         element, a translation initiation element, a truncated fragment         III of a coding element, and an intron fragment IV;     -   where, a 3′ end of the truncated fragment III of the coding         element includes a ribozyme recognition site IV that consists of         a second predetermined number of nucleotides located at the 3′         end of the truncated fragment III of coding element;     -   a 5′ end of the truncated fragment IV of the coding element         includes a ribozyme recognition site III that consists of a         first predetermined number of nucleotides located at the 5′ end         of the truncated fragment IV of coding element;     -   the nucleotide sequence of the truncated fragment III of the         coding element and the nucleotide sequence of the truncated         fragment IV of the coding element form a coding element sequence         encoding at least one target polypeptide in the 5′ to 3′         direction; the nucleotide sequence of the truncated fragment III         of the coding element corresponds to a partial sequence close to         the 5′ direction in the coding element sequence, and the         nucleotide sequence of the truncated fragment IV of the coding         element corresponds to a remaining partial sequence close to the         3′ direction in the coding element sequence;     -   where, the sequence of the intron fragment III is a reverse         sequence or a reverse complementary sequence of the nucleotide         sequence of the intron fragment I, and the sequence of intron         fragment IV is a reverse sequence or a reverse complementary         sequence of the nucleotide sequence of the intron fragment II;         the sequence of the ribozyme recognition site III is a reverse         sequence of the nucleotide sequence of the ribozyme recognition         site I, and the sequence of the ribozyme recognition site IV is         a reverse sequence of the nucleotide sequence of the ribozyme         recognition site II.

(2) The recombinant nucleic acid molecule according to (1), where the intron fragment I and the intron fragment II are derived from Group I intron, the ribozyme recognition site I is derived from a natural exon sequence ligated to the 5′ end of the intron fragment I, and the ribozyme recognition site II is derived from a natural exon sequence ligated to the 3′ end of the intron fragment II; and

-   -   optionally, the Group I intron is derived from any one of the         following Group I introns: phage T4 td gene, Anabaena         tRNA^(Leu), TpaCOX2, and Ptu.

(3) The recombinant nucleic acid molecule according to (1) or (2), where the first predetermined number of nucleotides is selected from 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to 10 nucleotides.

(4) The recombinant nucleic acid molecule according to any one of (1) to (3), where the second predetermined number of nucleotides is selected from 1 to 100 nucleotides, preferably 1 to 50 nucleotides, and more preferably 1 to 10 nucleotides.

(5) The recombinant nucleic acid molecule according to any one of (1) to (4), where a sum of the first predetermined number and the second predetermined number is not equal to 3y, where y≥1 and y is an integer.

(6) The recombinant nucleic acid molecule according to any one of (1) to (5), where the translation initiation element includes a sequence with an activity of initiating translation of an editing region; and

-   -   optionally, the sequence with the activity of initiating         translation of an editing region is selected from one of or a         combination of two or more of: an IRES sequence, a 5′ UTR         sequence, a Kozak sequence, a sequence with m⁶A modification,         and a complementary sequence of ribosome 18S rRNA.

(7) The recombinant nucleic acid molecule according to any one of (1) to (6), where the recombinant nucleic acid molecule is used for preparing a circular RNA including a coding element, where the coding element in the circular RNA includes a coding region 1, optionally (a) at least one coding region 2, and optionally (b) at least one coding region 3;

-   -   the truncated fragment I of the coding element and the truncated         fragment II of the coding element form the coding region 1,         optionally (a) the at least one coding region 2, and         optionally (b) the at least one coding region 3; the recombinant         nucleic acid molecule includes elements arranged in the order         shown in any one of (i) to (iv) in the 5′ to 3′ direction:     -   (i) the intron fragment II, the truncated fragment II of the         coding region 1, the at least one coding region 2, the         translation initiation element, the truncated fragment I of the         coding region 1, and the intron fragment II;     -   (ii) the intron fragment II, the truncated fragment II of the         coding region 1, the translation initiation element, the at         least one coding region 3, the truncated fragment I of coding         region 1, and the intron fragment I;     -   (iii) the intron fragment II, the truncated fragment II of the         coding region 1, the at least one coding region 2, the         translation initiation element, the at least one coding region         3, the truncated fragment I of the coding region 1, and the         intron fragment I;     -   (iv) the intron fragment II, the truncated fragment II of the         coding region 1, the translation initiation element, the         truncated fragment I of the coding region 1, and the intron         fragment I;     -   alternatively, the truncated fragment III of the coding element         and the truncated fragment IV of the coding element form the         coding region 1, optionally (a) the at least one coding region         2, and optionally (b) the at least one coding region 3; the         recombinant nucleic acid molecule includes elements arranged in         the order shown in any one of (v) to (viii) in the 5′ to 3′         direction:     -   (v) the intron fragment III, the truncated fragment IV of the         coding region 1, the at least one coding region 2, the         translation initiation element, the truncated fragment III of         the coding region 1, and the intron fragment IV;     -   (vi) the intron fragment III, the truncated fragment IV of the         coding region 1, the translation initiation element, the at         least one coding region 3, the truncated fragment III of the         coding region 1, and the intron fragment IV;     -   (vii) the intron fragment III, the truncated fragment IV of the         coding region 1, the at least one coding region 2, the         translation initiation element, the at least one coding region         3, the truncated fragment III of the coding region 1, and the         intron fragment IV;     -   (viii) the intron fragment III, the truncated fragment IV of the         coding region 1, the translation initiation element, the         truncated fragment III of the coding region 1, and the intron         fragment IV;     -   where, the coding region 1, each coding region 2 and each coding         region 3 encode any type of target polypeptides independently of         one another.

(8) The recombinant nucleic acid molecule according to (7), where the recombinant nucleic acid molecule includes one or both elements of (i) to (ii):

-   -   (i) a linker located between the truncated fragment II of the         coding region 1, and the coding region 2;     -   (ii) a linker located between the coding region 3 and the         truncated fragment I of the coding region 1;     -   alternatively, the recombinant nucleic acid molecule includes         one or both elements of (iii) to (iv):     -   (iii) a linker located between the truncated fragment IV of the         coding region 1, and the coding region 2;     -   (iv) a linker located between the coding region 3 and the         truncated fragment III of the coding region 1;     -   optionally, the coding region 2 has a quantity of at least 2,         and the recombinant nucleic acid molecule includes a linker         located between any two coding regions 2;     -   optionally, the coding region 3 has a quantity of at least 2,         and the recombinant nucleic acid molecule includes a linker         located between any two coding regions 3; and     -   preferably, the linker is a 2A peptide-encoding polynucleotide.

(9) The recombinant nucleic acid molecule according to any one of (1) to (6), where the recombinant nucleic acid molecule is used for preparing a circular RNA including a coding element; where the coding element in the circular RNA includes a coding region 1, at least one coding region 4, and a translation initiation element located between any two adjacent coding regions;

-   -   the truncated fragment I of the coding element and the truncated         fragment II of the coding element form the coding region 1, the         at least one coding region 4, and the translation initiation         element located between any two adjacent coding regions; or,     -   the truncated fragment III of the coding element and the         truncated fragment IV of the coding element form the coding         region 1, the at least one coding region 4, and the translation         initiation element located between any two adjacent coding         regions.

(10) The recombinant nucleic acid molecule according to any one of (1) to (9), where the target polypeptide is a human-derived protein or non-human derived protein; and

-   -   optionally, the target polypeptide is selected from one of or a         combination of two or more of: an antigen, an antibody, an         antigen-binding fragment, a fluorescent protein, a protein with         therapeutic activity, and a protein with gene-editing activity.

(11) The recombinant nucleic acid molecule according to any one of (1) to (10), where the recombinant nucleic acid molecule further includes an insertion element between the truncated fragment II and the translation initiation element, or an insertion element between the truncated fragment IV and the translation initiation element; the insertion element is at least one selected from the group consisting of (i) to (iii):

-   -   (i) a transcriptional regulatory element, (ii) a translational         regulatory element, and (iii) a purification element;     -   preferably, the insertion element is ligated to the 5′ end of         any translation initiation element;     -   optionally, the insertion element comprises a sequence selected         from one of or a combination of two or more of:     -   an untranslated region sequence, a polyA sequence, an aptamer         sequence, a riboswitch sequence, and a transcriptional         regulator-binding sequence.

(12) The recombinant nucleic acid molecule according to any one of (1) to (11), where the recombinant nucleic acid molecule further includes a 5′ homologous arm and a 3′ homologous arm, and the nucleotide sequence of the 5′ homologous arm is hybridized with the nucleotide sequence of the 3′ homologous arm;

-   -   the 5′ homologous arm is ligated to the 5′ end of the intron         fragment II, and the 3′ homologous arm is ligated to the 3′ end         of the intron fragment I; or, the 5′ homologous arm is ligated         to the 5′ end of the intron fragment III, and the 3′ homologous         arm is ligated to the 3′ end of the 3′ intron fragment IV.

(13) The recombinant nucleic acid molecule according to any one of (1) to (12), where a nucleotide sequence derived from an exon is not included in any one of the intron fragment I, the translation initiation element, or the intron fragment II, or between any two of the intron fragment I, the truncated fragment II of the coding element, the translation initiation element, the truncated fragment I of the coding element, and the intron fragment II; or,

-   -   a reverse sequence or reverse complementary sequence of a         nucleotide sequence derived from an exon is not included in any         one of the intron fragment III, the translation initiation         element, and the intron fragment IV, or between any two of the         intron fragment III, the truncated fragment IV of the coding         element, the translation initiation element, the truncated         fragment III of the coding element, and the intron fragment IV.

(14) A recombinant expression vector, including the recombinant nucleic acid molecule according to any one of (1) to (13).

(15) Use of the recombinant nucleic acid molecule according to any one of (1) to (13) or the recombinant expression vector according to (14) for preparing a circular RNA in vitro.

(16) A method for preparing a circular RNA in vitro, including the steps of:

-   -   transcribing the recombinant nucleic acid molecule according to         any one of (1) to (13) or the recombinant expression vector         according to (14) to form a cyclization precursor nucleic acid         molecule;     -   cyclizing the cyclization precursor nucleic acid to obtain the         circular RNA; and     -   optionally, further including the step of purifying the circular         RNA.

(17) A circular RNA prepared by the recombinant nucleic acid molecule according to any one of (1) to (13), the recombinant expression vector according to (14), or the method according to (16).

(18) A circular RNA, including elements arranged in the following order in the 5′ to 3′ direction:

-   -   a translation initiation element, a coding element for encoding         at least one target polypeptide;     -   optionally, the circular RNA includes an insertion element         located between the 5′ end of the translation initiation element         and the 3′ end of the coding element;     -   optionally, the translation initiation element, the target         polypeptide or the insertion element are defined according to         any one of (6) and (10) to (11).

(19) The circular RNA according to (18), where the coding element of the circular RNA includes a coding region 1, and at least one in the group consisting of (i) to (ii):

-   -   (i) at least one coding region 2, and (ii) at least one coding         region 3; where each coding region encodes any type of target         polypeptide independently of one another;     -   preferably, any two adjacent coding regions are linked by a         linker.

(20) The circular RNA according to (18), where the coding element of the circular RNA includes the coding region 1 and at least one coding region 4, and the 5′ end of any coding region is ligated to a translation initiation element.

(21) The circular RNA according to any one of (18) to (20), where the insertion element is ligated to the 5′ end of any translation initiation element.

(22) A composition including the recombinant nucleic acid molecule according to any one of (1) to (13), the recombinant expression vector according to (14), or the circular RNA according to any one of (17) to (21); preferably the circular RNA according to any one of (17) to (21);

-   -   optionally, the composition further includes one or more         pharmaceutically acceptable carriers; and     -   optionally, the pharmaceutically acceptable carrier is selected         from a lipid, a polymer or a lipid-polymer complex.

(23) A method for expressing a target polypeptide in a cell, where the method includes the step of delivering the circular RNA according to any one of (17) to (21) or the composition according to (18) into the cell.

(24) A method for preventing or treating diseases, where the method includes administering the circular RNA according to any one of (17) to (21) or the composition according to (22) to a subject.

(25) A method for screening a target coding region sequence including a ribozyme recognition site, where the coding region sequence is a coding sequence of a target polypeptide; the method includes the following steps:

-   -   S1, extracting m amino acid units from the target polypeptide         including q amino acids in the N-terminal-to-C-terminal         direction, with each of the amino acid units including n amino         acids; where at least one repeated amino acid is included         between any two adjacent amino acid units, n is an integer and         n≥2, m is an integer and m≥1; preferably, m=q+1-n;     -   S2, determining m codon sequence sets, where each of the codon         sequence sets includes codon sequences corresponding to each of         the amino acid units;     -   S3, traversing the m codon sequence sets to obtain a matching         value of each codon sequence in each of the codon sequence sets         with a target motif;     -   S4, determining a target codon sequence in the codon sequence         sets based on the matching value, where the position of the         target codon sequence corresponding to the coding region         sequence is the implantation position of the ribozyme         recognition site, and the coding region sequence including the         target codon sequence at the implantation position is the target         coding region sequence including the ribozyme recognition site.

(26) The method according to (25), where the target motif includes a ribozyme recognition site sequence that is formed by ligating the nucleotide sequence of a ribozyme recognition site I to the nucleotide sequence of a ribozyme recognition site II; or is formed by ligating the nucleotide sequence of a ribozyme recognition site III to the nucleotide sequence of a ribozyme recognition site IV;

-   -   at least one of the 5′ end and the 3′ end of the ribozyme         recognition site sequence is ligated to x nucleotide to obtain a         target motif with 3n nucleotide; where each x is an integer≥0         independently of one another, and each ligated nucleotide is         selected from any type of nucleotide independently of one         another.

(27) The method according to (25) or (26), where the target motif includes an effective base pair that includes two bases at the linking position of ribozyme recognition site I and ribozyme recognition site II; the obtaining a matching value of each codon sequence in each of the codon sequence sets with a target motif includes:

-   -   determining whether the base at a position corresponding to the         effective base pair in the codon sequence is an effective base,         and if the codon sequence does not include an effective base         pair, disabling output of an alignment value of the codon         sequence;     -   if the codon sequence includes an effective base pair,         determining the alignment value between each base in each codon         sequence with the corresponding base in the target motif in the         5′ to 3′ direction; and     -   obtaining the matching value of each codon sequence with the         target motif based on the alignment value of each base in each         codon sequence.

(28) The method according to (27), where the obtaining a matching value of each codon sequence in each of the codon sequence sets with a target motif further includes:

-   -   determining whether each codon sequence in each of the codon         sequence sets is hybridized with an intron sequence to obtain a         complementary value of each codon sequence in each of the codon         sequence sets;     -   determining the matching value of each codon sequence in each of         the codon sequence sets with the target motif based on the         alignment value and the complementary value.

(29) A screening system for screening a target coding region sequence including a ribozyme recognition site, where the coding region sequence is a nucleotide sequence encoding a target polypeptide;

-   -   the screening system includes:     -   a target motif constructing module configured to ligate x         nucleotide to at least one of the 5′ end and the 3′ end of the         ribozyme recognition site sequence to obtain a target motif with         3n nucleotide; where each x is an integer≥0 independently of one         another, and each ligated nucleotide is selected from any type         of nucleotide independently of one another;     -   an amino acid unit extraction module configured to extract m         amino acid units from the target polypeptide including q amino         acids in the N-terminal-to-C-terminal direction, with each of         the amino acid units including n amino acids; where at least one         repeated amino acid is included between any two adjacent amino         acid units, n is an integer and n≥2, m is an integer and m≥1;         preferably, m=q+1−n;     -   a codon sequence set extraction module configured to determine m         codon sequence sets, where each of the codon sequence sets         includes codon sequences corresponding to each of the amino acid         units;     -   a matching value calculation module configured to traverse the m         codon sequence sets to obtain a matching value of each codon         sequence in each of the codon sequence sets with a target motif;     -   a target codon sequence screening module configured to determine         a target codon sequence in the codon sequence sets based on the         matching value, where the position of the target codon sequence         corresponding to the coding region sequence is the implantation         position of the ribozyme recognition site, and the coding region         sequence including the target codon sequence at the implantation         position is the target coding region sequence comprising the         ribozyme recognition site;     -   preferably, the target motif includes a ribozyme recognition         site sequence that is formed by ligating the nucleotide sequence         of a ribozyme recognition site I to the nucleotide sequence of a         ribozyme recognition site II; or is formed by ligating the         nucleotide sequence of a ribozyme recognition site III to the         nucleotide sequence of a ribozyme recognition site IV; and     -   at least one of the 5′ end and the 3′ end of the ribozyme         recognition site sequence is ligated to x nucleotide to obtain         the target motif with 3n nucleotide; where each x is an         integer≥0 independently of one another, and each ligated         nucleotide is selected from any type of nucleotide independently         of one another.

(30) The screening system according to (29), where the target motif includes an effective base pair that corresponds to two bases at the linking position of the ribozyme recognition site I and the ribozyme recognition site II; where the matching value calculation module includes:

-   -   an effective base pair determination unit configured to         determine whether the base at a position corresponding to the         effective base pair in the codon sequence is an effective base,         and if the codon sequence does not includes the effective base         pair, disabling output of the alignment value of the codon         sequence;     -   an alignment value determination unit configured to determine         the alignment value between each base in each codon sequence         with the corresponding base in the target motif in the 5′ to 3′         direction;     -   a matching value output unit configured to obtain the matching         value of each codon sequence with the target motif based on the         alignment value of each base in each codon sequence.

(31) The screening system according to (30), where the matching value calculation module further includes:

-   -   a complementary value calculation module configured to determine         whether each codon sequence in each of the codon sequence sets         is hybridized with an intron sequence to obtain a complementary         value of each codon sequence in each of the codon sequence sets;     -   the matching value output unit configured to determine the         matching value of each codon sequence in each of the codon         sequence sets with the target motif based on the alignment value         and the complementary value.

(32) A method for screening a ribozyme recognition site sequence, where the method includes:

-   -   determining a sequence to be screened, and the sequence to be         screened includes an intron sequence derived from Group I         intron, a first exon sequence ligated to the 5′ end of the         intron sequence, and a second exon sequence ligated to the 3′         end of the intron sequence;     -   obtaining a predicted RNA secondary structure based on the         sequence to be screened;     -   obtaining a ribozyme recognition site I with ribozyme         recognition activity in the first exon sequence and a ribozyme         recognition site II with ribozyme recognition activity in the         second exon sequence based on the predicted RNA secondary         structure;     -   determining the ribozyme recognition site sequence based on the         nucleotide sequence of the ribozyme recognition site I and the         nucleotide sequence of the ribozyme recognition site II;     -   optionally, the ribozyme recognition site sequence includes at         least one in the group consisting of (i) to (iv):     -   (i) the nucleotide sequence of the ribozyme recognition site I,     -   (i) the nucleotide sequence of the ribozyme recognition site II,     -   (iii) a mutant sequence of the ribozyme recognition site I with         ribozyme recognition activity,     -   (iv) a mutant sequence of the ribozyme recognition site II with         ribozyme recognition activity,     -   preferably, the ribozyme recognition site sequence is formed by         ligating any one of (i) and (iii) to any one of (ii) and (iv).

(33) The method according to (32), where the nucleotide sequence of the ribozyme recognition site I is hybridized with a leader sequence in the intron sequence, or the nucleotide sequence of the ribozyme recognition site II is hybridized with a leader sequence in the intron sequence.

(34) The method according to (32) or (33), where the method includes:

-   -   sequentially replacing the base on the ribozyme recognition site         I to obtain the mutant sequence of the ribozyme recognition site         I with ribozyme recognition activity, or     -   sequentially replacing the base on the ribozyme recognition site         II to obtain the mutant sequence of the ribozyme recognition         site II with ribozyme recognition activity.

Effects of Disclosure

In some embodiments, the recombinant nucleic acid molecule for preparing a circular RNA provided by the present disclosure includes: an intron fragment II, a truncated fragment II of a coding element, a translation initiation element, a truncated fragment I of a coding element, and an intron fragment I. The circular RNA is prepared by the recombinant nucleic acid molecule in vitro, in which under the guidance of the intron sequence, a linear nucleic acid molecules are ligated to form the circular RNA through the successive cleavage occurred at a splice site at the 3′ end of the truncated fragment I of a coding element and a splice site at the 5′ end of the truncated fragment II of a coding element, and a coding element for encoding at least one target polypeptide is formed by ligating the truncated fragment I of the coding element and the truncated fragment II of the coding element. In addition, as the ribozyme recognition site I and the ribozyme recognition site II are formed inside the truncated fragments of the coding element, no additional exon sequence is required to be introduced into the recombinant nucleic acid molecule, thus excluding the additional exon sequence from the circular RNA, thereby improving the sequence accuracy of the circular RNA molecule.

The recombinant nucleic acid molecule of the present disclosure provides a Clean PIE system with a novel structure for preparing a circular RNA in vitro. As compared to a classic PIE system, the Clean PIE system provided by the present disclosure is capable of improving the sequence accuracy of the circular RNA molecule, reducing changes in the secondary structure of the circular RNA, and further reducing immunogenicity of the circular RNA, improving stability of the circular RNA in cells, reducing safety risk of circular RNAs in clinical application, and enabling large-scale production of circular RNAs in vitro, thus showing wide application prospects in the fields of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell (DC) tumor vaccines, mRNA-based gene therapy, protein supplement therapy, and the like.

In some embodiments, the recombinant nucleic acid molecule for preparing a circular RNA provided by the present disclosure includes: an intron fragment III, a truncated fragment IV of a coding element, a translation initiation element, a truncated fragment III of a coding element, and an intron fragment IV. In case that the recombinant nucleic acid molecule is used for preparing a circular RNA in vitro, under the guidance of the intron sequence, linear nucleic acid molecules are ligated to form the circular RNA through successive cleavage occurred at the splice site at the 5′ end of the truncated fragment IV of the coding element and the splice site at the 3′ end of the truncated fragment III of the coding element, and the truncated fragment III of the coding element and the truncated fragment IV of the coding element are ligated to form the coding element for encoding at least one target polypeptide. In addition, it avoids introduction of additional exon sequences into the circular RNA, which may improve sequence accuracy, reduce immunogenicity, improve stability, and reduce safety risks of clinical application thereof, and enabling large-scale production of the circular RNA in vitro, thus showing good application prospects in the fields of nucleic acid vaccines and gene therapy.

In some embodiments, as no spacer, homology arm, exon and other fragments are required to be introduced, the recombinant nucleic acid molecule for preparing circular RNAs provided by the present disclosure is structurally simple, such that the resulting circular RNA is safe and suitable for large-scale industrial production of circular RNAs in vitro.

In some embodiments, in the recombinant nucleic acid molecule for preparing a circular RNA provided by the present disclosure, translation initiation elements are selective from a variety of sequences, each of which allows for efficient translation of the coding region in a circular RNA, thus providing selectivity from a variety of sequences for preparation of a circular RNA.

In some embodiments, in the recombinant nucleic acid molecule for preparing a circular RNA provided by the present disclosure, an insertion element with different functions may be further introduced, such as a transcriptional regulatory element, a translational regulatory element or a purification element into the circular RNA, providing specific regulation of target polypeptides expressed by the circular RNA. In addition, the in vitro purification of circular RNA may specifically regulate the expression abundance of target polypeptides, improving the disease treatment effects of circular RNAs.

In some embodiments, the circular RNA provided by the present disclosure is prepared by using the recombinant nucleic acid molecule described above. With no additional exon sequences introduced, the circular RNA has a high sequence accuracy, small changes in the secondary structure, high biological safety and structural stability, and low immunogenicity, thus suitable for the field of clinical disease treatment.

In some embodiments, in the method for screening a target coding region sequence provided by the present disclosure, the target coding region sequence including a ribozyme recognition site is obtained by sorting the degenerate codon sequence of the amino acid of the target polypeptide, aligning it with the target motif, and scoring. The target coding region sequence may be truncated at the position of the ribozyme recognition site to obtain the fragment I of the coding element and the truncated fragment II of the coding element, or the truncated fragment III of the coding element and the truncated fragment IV of the coding element. The ribozyme recognition site is inserted in the truncated fragments of the coding element, which avoids introduction of additional exon sequences into the circular RNA. The method for screening a target coding region sequence provided by the present disclosure is suitable for integrating ribozyme recognition sites in any type of PIE system into the coding region sequence, showing wide application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of secondary structures of circular RNAs ligated and formed with T4 ligase, T4 td PIE system, and Anabaena PIE system respectively, in the cited reference [14].

FIGS. 2A through 2C show schematic structural diagrams of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 2D through 2F show schematic structural diagrams of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 3A and 3B show a schematic structural diagram of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 4A through 4D show schematic structural diagrams of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 5A through 5D show schematic structural diagrams of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 6A through 6D show a schematic structural diagram of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 7A through 7C shows a schematic structural diagram of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 7D through 7F shows a schematic structural diagram of a recombinant nucleic acid molecule for preparing a circRNA according to the present disclosure (a Clean PIE system).

FIGS. 8A and 8B shows circRNA structures, wherein A shows circRNA structure prepared by the Clean PIE system of the present disclosure, and B shows circRNA structure prepared by the classic PIE system.

FIGS. 9A and 9B show circRNA structures prepared by the Clean PIE system of the present disclosure.

FIGS. 10A through 10C show circRNA structures prepared by the Clean PIE system of the present disclosure.

FIGS. 11A and 11B shows circRNA structure prepared by the Clean PIE system of the present disclosure.

FIG. 12 shows a schematic procedure diagram of forming a circRNA by circularization of a classic PIE system.

FIG. 13 shows a prognostic diagram of a secondary structure derived from a T4td intron.

FIG. 14 shows a prognostic diagram of a secondary structure derived from a TpaCOX2 intron.

FIG. 15 shows a prognostic diagram of a secondary structure derived from a Ptu intron.

FIG. 16 shows scoring criteria for screening a target coding fragment sequence including a ribozyme recognition site, by taking a T4td PIE as an example.

FIG. 17 shows an automated flow chart for determining a target including a ribozyme recognition site by using a screening system.

FIG. 18 shows detection results of agarose gel electrophoresis of the digested products (A) of the plasmids used for the preparation of circular mRNA by using a classic PIE system and a Clean PIE system of the present disclosure respectively, and that of the circularized products (B).

FIGS. 19A and 19B show detection results of in vitro expression level of circular mRNAs prepared by a classic PIE system, and a Clean PIE system of the present disclosure, respectively.

FIG. 20 shows detection results of agarose gel electrophoresis of circular mRNAs expressing different proteins prepared by circularization of a Clean PIE system.

FIG. 21 shows evaluation results of scoring a matching value for genes over 1000 bp and 500 bp in the genome of Escherichia coli.

FIG. 22 shows RNaseR digestion of linear and circular mRNAs produced by different PIE systems.

FIGS. 23A through 23C show analysis results of capillary electrophoresis.

FIGS. 24A and 24B show PCR sequencing analysis results of cDNA after reverse transcription of Fluc and IL12.

FIGS. 25A and 25B shows expression detection results of uncircularized linear mRNAs for preparing circular mRNAs respectively in a classic PIE system and a Clean PIE system of the present disclosure, and that of circularized circular mRNAs; where A shows structures of uncircularized linear mRNAs of the classic PIE system and the Clean PIE system, and B shows protein expression results of the linear mRNAs detected by western blotting.

FIG. 26 shows expression detection results of circular mRNAs prepared by a Clean PIE system after inserting a translational regulatory element (polyAC).

FIGS. 27A and 27B show tissue-specific expression of circular mRNAs obtained by circularization of a Clean PIE system and regulated by a translational regulatory element, where A shows the expression of circular mRNAs with miR122 site injected in mice, and B shows a frame structure of the Clean PIE system with miR122 site.

FIG. 28 shows detection results of gel electrophoresis of circular mRNAs purified by S1m RNA aptamer.

FIG. 29 shows expression of immune factors induced by circular mRNAs prepared by using a Clean PIE system (corresponding to the Clean PIE shown) and Anabaena PIE system (corresponding to the ana-PIE shown).

FIG. 30 shows detection results of gel electrophoresis of circular mRNAs prepared by the Clean PIE system with and without homology arms.

FIG. 31 shows analysis results of agarose gel electrophoresis of circular mRNAs constructed using truncated sequences of coding regions with different scores.

FIGS. 32A through 32C shows detection results of protein expression of eGFP and firefly Luciferase expressed by circular mRNAs containing different coding regions ligated in tandem by T2A.

FIG. 33A through 33C shows detection results of protein expression of eGFP and firefly Luciferase expressed by circular mRNAs containing different coding regions ligated in tandem by IRES.

FIG. 34 shows structural characteristics of Group I intron.

DETAILED DESCRIPTION Definitions

When used in conjunction with the terms “comprising”, “including” or “containing” in the claims and/or the specification, the word “a” or “an” may refer to “one”, but it may also refer to “one or more”, “at least one”, and “one or more than one”.

As used in the claims and the specification, the words “comprising”, “having”, “including” or “containing” are inclusive or open-ended, and do not exclude additional elements or method steps that are not cited.

Throughout this application, the term “about” is intended to refer to a value that includes standard deviations of errors of a device or method used to measure this value.

Although the term “or” as used in this disclosure may be defined just as alternatives and “and/or”, the term “or” in the claims refers to “and/or”, unless expressly defined only as alternatives or alternatives that are mutually exclusive.

The terms “polypeptide”, “peptide”, and “protein” as used herein are interchangeable and are amino acid polymers of any length. The polymer, linear or branched, may contain modified amino acids, and may be separated by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with labeling components.

As used herein, the “PIE system”, also known as permuted introns and exons, is a method for ligating to form a circular RNA by a self-splicing system of Group I intron.

As used herein, the “Group I intron” has a self-splicing system for circularization in the presence of GTP and Mg²⁺.

Group I introns are a class of huge ribozymes capable of self-splicing, which are widely found in many species, mainly being involved in catalytic excision of precursors of mRNA, tRNA and rRNA. The core secondary structure thereof typically includes nine pairing regions (P1-P9) and corresponding loop regions (L1-L9) (FIG. 34 ). Splicing of Group I introns is processed by two sequential transesterification reactions. The exogenous guanosine or guanosine nucleotide (G) first docks onto the active G-binding site located in P7, and its 3′-OH is aligned to attack the phosphodiester bond at the 5′ splice site located in P1, resulting in a free 3′-OH group at the upstream exon and the exogenous G being attached to the 5′ end of the intron. Then the terminal G (omega G) of the intron swaps the exogenous G and occupies the G-binding site to organize the second ester-transfer reaction: the 3′-OH group of the upstream exon in P1 is aligned to attack the 3′ splice site in P10, leading to the ligation of the adjacent upstream and downstream exons and release of the catalytic intron. Furthermore, the sequence of the segment joining P6 and P7 is J6/7 sequence, and the sequence of the segment joining P8 and P7 is J8/7 sequence. The Group I intron typically includes structural characteristics as shown in FIG. 34 (from Burke J M, Belfort M, Cech T R, et al. Structural Conventions for Group I introns [J]. Nucleic Acids Research, 1987, 15 (18): 7217-7221).

As used herein, an “internal guide sequence” typically refers to the nucleotide sequence of a fragment of Group I intron that is mutually paired with a corresponding exon sequence by Watson-Crick pairing or wobble pairing, generally in a P1 stem in a Group I intron.

As used herein, a “ribozyme” is intended to refer to RNA with catalytic activity. In some embodiments, a “ribozyme recognition site” herein refers to a polynucleotide sequence that may be recognized by the ribozyme and an internal broken of a phosphodiester bond can by occurred when RNA forms a ribozyme molecule with catalytic function.

As used herein, the term “circular nucleic acid molecule” refers to a nucleic acid molecule with a closed loop. In some embodiments, the circular nucleic acid molecule is a circular RNA molecule. More specifically, the circular nucleic acid molecule is a circular mRNA molecule.

As used herein, the term “linear RNA” refers to a circular RNA precursor capable of forming a circular RNA through a circularization reaction, which is typically formed by transcription of a linear DNA molecule (for example, vectors containing recombinant nucleic acid molecules, and the like).

As used herein, the term “IRES”, also known as “internal ribosome entry site”, is a translation control sequence, generally located at the 5′ end of a gene of interest, and enables translation of RNA in a cap-independent manner. The transcribed IRES may directly bind to a ribosome subunit, such that a start codon of mRNA is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5′ UTR of mRNA (immediately upstream of the start codon). The IRES functionally replaces the need for various protein factors that interact with a translation mechanism of eukaryotes.

As used herein, the term “translation initiation element” refers to any sequence element that may recruit ribosomes and initiate a translation process of RNA molecules. Illustratively, translation initiation elements include an IRES element, an m⁶A-modified sequence, or an initiation sequence for rolling circle translation, and the like.

The terms “coding region”, “protein coding region”, and “open reading frame (ORF)” may be used interchangeably herein. A coding region starts with a start codon, with consecutive nucleotide sequences with protein-encoding potency. In some embodiments, the coding region ends with a stop codon. In other embodiments, the coding region may not contain the stop codon.

The term “coding element” herein is formed in a circular RNA prepared by using a Clean PIE system (e.g., a recombinant nucleic acid molecule, a recombinant expression vector, or the like) of the present disclosure, and is used to encode at least one target polypeptide. Therefore, the coding element contains at least one coding region. Illustratively, coding elements contain 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more coding regions (including any integer value between any two numbers above). Moreover, a ribozyme recognition site is provided inside any one or more coding regions.

The “ribozyme recognition site” herein consists of a ribozyme recognition site I and a ribozyme recognition site II. It should be noted that the ribozyme recognition site is arranged within a coding region contained in the coding element. Accordingly, the ribozyme recognition site I and ribozyme recognition site II are only formed within the coding region contained in the coding element. Alternatively, the ribozyme recognition site consists of a ribozyme recognition site III and a ribozyme recognition site IV. It should be noted that the ribozyme recognition site is arranged within a coding region contained in the coding element. Accordingly, the ribozyme recognition site III and ribozyme recognition site IV are only formed inside the coding region contained in the coding element.

The nucleotide sequence of the truncated fragment I of the coding element and the nucleotide sequence of the truncated fragment II of the coding element of the present disclosure are formed by truncation of the coding element sequence, with the former one corresponding to a partial sequence close to the 5′ direction in the coding element sequence and the latter one corresponding to a remaining partial sequence close to the 3′ direction in the coding element sequence. Moreover, the ribozyme recognition site I and ribozyme recognition site II are located inside any coding region contained in the coding element.

Alternatively, the nucleotide sequence of the truncated fragment III of the coding element and the nucleotide sequence of the truncated fragment IV of the coding element herein are formed by truncation of the coding element sequence, with the former one corresponding to a partial sequence close to the 5′ direction in the coding element sequence and the latter one corresponding to a remaining partial sequence close to the 3′ direction in the coding element sequence. Moreover, the ribozyme recognition site III and ribozyme recognition site IV are located inside any coding region contained in the coding element.

Furthermore, in the case that two or more coding regions are contained in the coding element, the two adjacent coding regions may be linked by a linker or a translation initiation element. Accordingly, a linker linking to adjacent coding region sequences, or a translation initiation element linking to adjacent coding region sequence, is further contained inside at least one of the truncated fragments of coding element.

Illustratively, the coding element consists of one coding region 1 that contains a ribozyme recognition site. Accordingly, the truncated fragment I of the coding element is a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element is a truncated fragment II of coding region 1. Alternatively, the truncated fragment III of the coding element is a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element is a truncated fragment IV of the coding region 1.

Illustratively, the coding element includes a coding region 1 and a coding region 2 sequentially arranged in the 5′ to 3′ direction, and a ribozyme recognition site is provided inside the coding region 1. The coding region 1 is truncated to obtain a truncated fragment I of the coding element and a truncated fragment II of the coding element, or to obtain a truncated fragment III of the coding element and a truncated fragment IV of the coding element. Wherein, the truncated fragment I of the coding element is a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element includes a truncated fragment II of the coding region 1, and the coding region 2. Alternatively, the truncated fragment III of the coding element is a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element includes a truncated fragment IV of the coding region 1, and the coding region 2.

In some alternative embodiments, the truncated fragment II of the coding element further includes a linker located between the truncated fragment II of the coding region 1, and the coding region 2. Alternatively, the truncated fragment IV of the coding element further includes a linker located between truncated fragment IV of the coding region 1 and coding region 2.

Illustratively, the coding element includes a coding region 3 and a coding region 1 sequentially arranged in the 5′ to 3′ direction, and a ribozyme recognition site is provided inside the coding region 1. The coding region 1 is truncated to obtain a truncated fragment I of the coding element and a truncated fragment II of the coding element, or to obtain a truncated fragment III of the coding element and a truncated fragment IV of the coding element. The truncated fragment I of the coding element includes the coding region 3 and a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element is a truncated fragment II of the coding region 1. Alternatively, the truncated fragment III of the coding element includes the coding region 3 and a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element is a truncated fragment IV of the coding region 1.

In some alternative embodiments, the truncated fragment of the coding element further includes a linker located between the coding region 3 and the truncated fragment I of the coding region 1. Alternatively, the truncated fragment III of the coding element further includes a linker located between the coding region 3 and the truncated fragment III of the coding region 1.

As an example, the coding element includes a coding region 3, a coding region 1 and a coding region 2 sequentially arranged in the 5′ to 3′ direction, and a ribozyme recognition site is provided inside the coding region 1. The coding region 1 is truncated to obtain a truncated fragment I of the coding element and a truncated fragment II of the coding element, or to obtain a truncated fragment III of the coding element and a truncated fragment IV of the coding element. The truncated fragment I of the coding element includes the coding region 3 and a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element includes a truncated fragment II of the coding region 1 and the coding region 2. Alternatively, the truncated fragment III of the coding element includes the coding region 3 and a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element includes a truncated fragment IV of coding region 1, and the coding region 2.

In some alternative embodiments, the truncated fragment I of the coding element further includes a linker located between the coding region 3 and the truncated fragment I of the coding region 1. Alternatively, the truncated fragment III of the coding element further includes a linker located between the coding region 3 and the truncated fragment III of the coding region 1. In some alternative embodiments, the truncated fragment II of the coding element further includes a linker located between the truncated fragment II of the coding region 1, and the coding region 2. Alternatively, the truncated fragment IV of the coding element further includes a linker located between the truncated fragment IV of the coding region 1 and the coding region 2.

Illustratively, the coding element includes a coding region 1, a translation initiation element and a coding region 4 sequentially arranged in the 5′ to 3′ direction, and a ribozyme recognition site is provided inside the coding region 1. The coding region 1 is truncated to obtain a truncated fragment I of the coding element and a truncated fragment II of the coding element, or to obtain a truncated fragment III of the coding element and a truncated fragment IV of the coding element. The truncated fragment I of the coding element includes a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element includes a truncated fragment II of the coding region 1, the translation initiation element, and the coding region 4. The truncated fragment III of the coding element includes a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element includes a truncated fragment IV of the coding region 1, the translation initiation element, and the coding region 4.

Illustratively, the coding element includes a coding region 4, a translation initiation element and a coding region 1 sequentially arranged in the 5′ to 3′ direction, and a ribozyme recognition site is provided inside the coding region 1. The coding region 1 is truncated to obtain a truncated fragment I of the coding element and a truncated fragment II of the coding element, or to obtain a truncated fragment III of the coding element and a truncated fragment IV of the coding element. The truncated fragment I of the coding element includes the coding region 4, the translation initiation element and a truncated fragment I of the coding region 1, and the truncated fragment II of the coding element is a truncated fragment II of the coding region 1. The truncated fragment III of the coding element includes the coding region 4, the translation initiation element and a truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element is a truncated fragment IV of the coding region 1.

It should be noted that the number of the coding regions 2, 3 or 4 may be one, or two or more, which is not exhaustive herein.

The term “expression” includes any step involving production of polypeptides, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term “antibody”, as used herein in the broadest sense thereof, refers to a protein containing antigen-binding sites, encompassing natural antibodies and artificial antibodies with various structures, including but not limited to polyclonal, monoclonal, monospecific, multi specific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term “antibody” further includes an antibody fragment, such as Fab, F(ab′)₂, FV, scFv, Fd, dAb and another antibody fragment that retains antigen-binding capacity. In general, such fragment may include an antigen-binding fragment.

As used herein, the term “hybridization” refers to a process in which a base on one nucleic acid chain is combined with a complementary base on another nucleic acid chain by base-pairing. The hybridization reaction may be selective, allowing for selection of a specific target sequence even at low concentrations from a sample. The stringency of hybridization conditions (such as highly stringent, moderately stringent, and stringent conditions) may be adjusted by, for example, a concentration of a salt or formamide in a pre-hybridized solution and a hybridized solution, or a hybridization temperature, and the like. As an example, the stringency may be increased by decreasing the salt concentration, increasing the formamide concentration, or increasing the hybridization temperature. Generally, the stringent conditions include hybridization in at least about 0% to at least about 15% v/v formamide and at least about 1 M to at least about 2 M salt at a temperature of about 25° C. to about 42° C., and washing in at least about 1 M to at least about 2 M salt. The moderately stringent conditions include hybridization in at least about 16% to at least about 30% v/v formamide and at least about 0.5 M to at least about 0.9 M salt at a temperature of about 25° C. to about 65° C., and washing in at least about 0.5 M to at least about 0.9 M salt. The highly stringent conditions include hybridization in at least about 31% to at least about 50% v/v formamide and at least about 0.01 M to at least about 0.15 M salt at a temperature of at least about 65° C., and washing in at least about 0.01 M to at least about 0.15 M salt. The formamide is optional in these hybridization conditions. Other suitable hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in Molecular Biology, 4^(th)ed., John Wiley & Sons (1999).

The term “pharmaceutically acceptable carrier” used in the context herein refers to auxiliary materials widely used in the field of pharmaceutical production. The carriers are used mainly to provide a pharmaceutical composition with use safety, stable properties and/or specific functions, and also to provide a method that allows active ingredients of a drug to be dissolved at a desired rate after being administered to a subject, or promotes effective absorption of the active ingredients in a subject administrated with the drug. A pharmaceutically acceptable carrier may be an inert filler, or a functional ingredient that provides a function for a pharmaceutical composition (such as stabilizing overall pH value of the composition or preventing degradation of active ingredients in the composition). Non-limiting examples of the pharmaceutically acceptable carriers include, but are not limited to, an adhesive, a suspending agent, an emulsifier, a diluent (or a filler), a granulating agent, a tackifier, a disintegrating agent, a lubricant, an anti-adhesion agent, a glidant, a wetting agent, a gelling agent, an absorption retarder, a dissolution inhibitor, an enhancer, an adsorbent, a buffer, a chelating agent, a preservative, a coloring agent, a flavoring agent, a sweetener, and the like.

As used herein, the term “complementary” or “hybridized” is intended to refer to a “polynucleotide” and “oligonucleotide” related to a base-pairing rule (interchangeable terms and refers to a nucleotide sequence). For example, the sequence “CAGT” is complementary to the sequence “GTCA”. The complementation or hybridization may be “partial” or “complete”. A “partial” complementation or hybridization means that one or more nucleic acid bases are mispaired according to a base-pairing rule, and a “total” or “complete” complementation or hybridization between nucleic acids means that each nucleic acid base is paired with another base by base-pairing. The degree of complementarity or hybridization of a nucleic acid chain has an important influence on hybridization efficiency and intensity of such nucleic acid chain. This is particularly important in an amplification reaction and a detection method depending on binding between nucleic acids.

The term “recombinant nucleic acid molecule” refers to a polynucleotide with sequences that are not ligated together in nature. The recombinant polynucleotide may be contained in a suitable vector, through which the recombinant polynucleotide may be delivered to a suitable host cell. Then the polynucleotide is expressed in the recombinant host cell to produce, for example, a “recombinant polypeptide”, a “recombinant protein”, a “fusion protein”, or the like.

The term “recombinant expression vector” refers to a DNA structure that expresses, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector may include, for example, i) a genetic element set involved in regulating gene expression, such as a promoter and an enhancer; ii) a structure or coding sequence for transcribing into mRNA and translating into a protein; and iii) a transcription subunit for appropriate transcription and translation of initiation and termination sequences. The recombinant expression vector is constructed in any suitable way. The nature of the vector is not critical, and any vector may be used, including a plasmid, a virus, a phage, and a transposon. A possible vector used for the present disclosure includes, but is not limited to, a chromosomal, a non-chromosomal and a synthetic DNA sequence, such as a viral plasmid, a bacterial plasmid, a phage DNA, a yeast plasmid, and a vector derived from combination of a plasmid and a phage DNA, and a DNA from a virus such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.

The term “host cell” refers to a cell into which an exogenous polynucleotide has been introduced, including progenies of such cells. The host cell includes a “transformant” and a “transformed cell”, including a primary transformed cell and a progeny derived therefrom. The host cell may be any type of cell system used to generate an antibody of the present disclosure, including an eukaryotic cell, such as a mammalian cell, an insect cell, and a yeast cell, and a prokaryotic cell, such as a Escherichia coli cell. The host cell includes a cultured cell, and also includes a cell in a transgenic animal, a transgenic plant or a cultured plant tissue or an animal tissue. The term “recombinant host cell” covers a host cell that is different from a parent cell after introduction of a recombinant nucleic acid molecule, a recombinant expression vector and a circular RNA, and the recombinant host cell is specifically produced by transformation. The host cells of the present disclosure may be a prokaryotic cell or an eukaryotic cell, as long as it may be used to introduce a recombinant nucleic acid molecule, a recombinant expression vector, a circular RNAs, and the like of the present disclosure.

As used herein, the term “individual”, “patient” or “subject” includes a mammal. The mammal includes, but is not limited to, a domestic animal (for example, cattle, sheep, cats, dogs, and horses), a primate (for example, human and non-human primates, such as monkeys), a rabbit, and a rodent (for example, mice and rats).

As used herein, the terms “transformation”, “transfection” and “transduction” have the meaning commonly understood by those skilled in the art, that is, a process introducing exogenous DNA into a host. The transformation, transfection and transduction methods include any method for introducing a nucleic acid into a cell, including but not limited to, electroporation, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, microinjection, polyethylene glycol (PEG)-based method, DEAE-dextran-based method, cationic liposome-based method, and lithium acetate-DMOS-based method.

As used herein, the “treatment” refers to alleviation of symptoms of a disease by contact (e.g., administration) of a circular RNA, a circularization precursor RNA, a composition, and the like of the present disclosure to a subject suffering from such disease, as compared to the scenario without such contact, and does not necessarily mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that some symptoms of disease occurred in a body.

As used herein, the “prevention” refers to alleviation of symptoms of a disease by contact (e.g., administration) of a circular RNA, a circularization precursor RNA, a composition, and the like of the present disclosure to a subject before suffering from such disease, as compared to the scenario without such contact, and does not necessarily mean that the symptoms of the disease must be completely suppressed.

As used herein, the term “effective amount” refers to such an amount or a dose of a recombinant nucleic acid molecule, a recombinant expression vector, a circularization precursor RNA, a circular RNA, a vaccine, or a composition of the present disclosure, that produces an expected effect in a patient in need of treatment or prevention after being administered in a single dose or in multiple doses. The effective amount may be easily determined by an attending physician as a skilled person in the art by considering the following factors: for example, species such as mammals; size, age and general health thereof; a related specific disease; a degree or severity of the disease; a response of an individual; a specific antibody administered; an administration mode; bioavailability characteristics of the administrated preparation; a selected dosage regimen; and an application of any concomitant therapy.

As used herein, the term “individual”, “patient” or “subject” includes a mammal. The mammal includes, but is not limited to, a domestic animal (for example, cattle, sheep, cats, dogs, and horses), a primate (for example, human and non-human primates, such as monkeys), a rabbit, and a rodent (for example, mice and rats).

Unless defined otherwise or clearly indicated in the context, all technical and scientific terms used herein have the same meaning as those commonly understood by a person of ordinary skill in the art to which the present disclosure belongs.

Clean PIE System

A process in which a classic PIE system is ligated to form a circular RNA is shown in FIGS. 5A through 5D, where the linear RNA includes the following elements ligated sequentially: a 3′ intron, a second exon E2 (Exon2), an exogenous fragment, a first exon E1 (Exon1), and a 5′ intron. With GTP and Mg²⁺ presenting in the environment, GTP attacks a linking position between the E1 and the 5′ intron, resulting in cleavage of a 5′ splice site (5ss) and release of the 5′ intron. The 3-OH end of E1 then attacks a linking position between the 3′ intron and the E2, resulting in cleavage of a 3′ splice site (3ss) and release of the 3′ intron. Finally, they are ligated to form a target circular RNA.

However, using a classic PIE system may lead to presence of additional exon sequences of E1 and E2 in the circular RNA, and reduced sequence accuracy of the circular RNAs, resulting in an increase of natural immunogenicity of the circular RNA, and susceptibility to degradation in cells.

To solve the problems above, the present disclosure provides a Clean PIE system with a novel structure, which may be used to prepare a circular RNA through self-splicing of the PIE system without changing a protein expression sequence, exhibiting high circularization efficiency. In addition, there is no need to introduce additional E1 and E2 sequences into the circularized circular RNA, which not only simplifies the structure of the circular RNA and reduces various potential safety hazards, but also improves the sequence accuracy of the circular RNA, reduces natural immunogenicity of the circular RNA, and improves stability thereof in cells, thus making it suitable for clinical application fields, such as serving as a gene therapy vector, an expressing therapeutic protein, serving as a nucleic acid vaccine and the like, showing broad application prospects.

The Clean PIE system of the present disclosure includes, but is not limited to, a DNA construct used to prepare a circular RNA, a recombinant expression vector containing the DNA construct, a circularization precursor RNA molecule obtained by in vitro transcription by using the recombinant expression vector, and the like.

In some embodiments, the present disclosure provides a recombinant nucleic acid molecule for preparing a circular RNA. Illustratively, the recombinant nucleic acid molecule may be the above-mentioned DNA construct for preparing a circular RNA, a circularization precursor RNA molecule, and the like.

In some embodiments, the structure of the recombinant nucleic acid molecule, as shown in FIG. 2A, includes elements arranged in the following order in the 5′ to 3′ direction: an intron fragment II, a truncated fragment II of a coding element, a translation initiation element, a truncated fragment I of a coding element, and an intron fragment I.

The 3′ end of the truncated fragment I of the coding element includes a ribozyme recognition site I that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment I of the coding element; the 5′ end of the truncated fragment II of the coding element contains a ribozyme recognition site II that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment II of the coding element.

The nucleotide sequence of the truncated fragment I of the coding element and the nucleotide sequence of the truncated fragment II of the coding element are used to form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction. The nucleotide sequence of the truncated fragment I of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence, and the nucleotide sequence of the truncated fragment II of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence.

It should be noted that the coding element is formed in the circular RNA prepared from the recombinant nucleic acid molecule. In addition, the coding element includes one or two or more coding regions encoding a target polypeptide. In the presence of two or more coding regions in the coding element, the coding element may also includes a linker located between two adjacent coding regions, a translation initiation element between two adjacent coding regions, or other types of sequences desired.

The nucleotide sequence of the intron fragment I and the nucleotide sequence of the intron fragment II are used to form an intron sequence in the 5′ to 3′ direction. The nucleotide sequence of the intron fragment I includes a partial sequence close to the 5′ direction in the intron sequence, and the nucleotide sequence of the intron fragment II includes a remaining sequence close to the 3′ direction in the intron sequence.

In other words, the nucleotide sequence of the truncated fragment I of the coding element and the nucleotide sequence of the truncated fragment II of the coding element may be ligated to form a coding element sequence encoding at least one target polypeptide, and the nucleotide sequence of the intron fragment I and the nucleotide sequence of the intron fragment II may be ligated to form an intron sequence. In preparing the circular RNAs by the recombinant nucleic acid molecule with the above structure, cleavage occurs at a linking position between a ribozyme recognition site I and the intron fragment I, releasing the intron fragment I. After that, cleavage occurs at a linking position of a ribozyme recognition site II and the intron fragment II, releasing the intron fragment II. The 3′ end of truncated fragment I of the coding element is ligated to the 5′ end of truncated fragment II of coding element to form a circular molecule. In the present disclosure, without changing the target polypeptide sequence encoded by the coding element and introducing additional E1 and E2 sequences, the circular RNA encoding the target protein is obtained by self-splicing, with a high sequence accuracy, stability, and low immunogenicity.

In other embodiments, a recombinant nucleic acid molecule, as shown in FIG. 2D, includes elements arranged in the following order in the 5′ to 3′ direction: an intron fragment III, a truncated fragment IV of a coding element, a translation initiation element, a truncated fragment III of a coding element, and an intron fragment IV.

The 3′ end of the truncated fragment III of the coding element includes a ribozyme recognition site IV that consists of a second predetermined number of nucleotides located at the 3′ end of the truncated fragment III of coding element. The 5′ end of the truncated fragment IV of the coding element contains a ribozyme recognition site III that consists of a first predetermined number of nucleotides located at the 5′ end of the truncated fragment IV of the coding element.

The nucleotide sequence of the truncated fragment III of the coding element and the nucleotide sequence of the truncated fragment IV of the coding element are used to form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction. The nucleotide sequence of the truncated fragment III of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence, and the nucleotide sequence of the truncated fragment IV of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence. The sequence of the intron fragment III is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment I, and the sequence of intron fragment IV is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment II. The sequence of the ribozyme recognition site III is a reverse sequence of the nucleotide sequence of the ribozyme recognition site I, and the sequence of the ribozyme recognition site IV is a reverse sequence of the nucleotide sequence of the ribozyme recognition site II.

As discovered by the present disclosure, the reverse sequence or reverse complementary sequence of intron sequence may also construct the Clean PIE system. In the present disclosure, the reverse sequence or reverse complementary sequence of a 5′ portion of the intron is used as the intron fragment III, and the reverse sequence or reverse complementary sequence of a 3′ portion of the intron is used as the intron fragment IV. The intron fragment III is ligated to the 5′ end of the truncated fragment IV of the coding element, corresponding to the intron fragment III, the first predetermined number of nucleotides at the 5′ end of truncated fragment IV of the coding element constitute the ribozyme recognition site III, and the sequence of the ribozyme recognition site III is a reverse sequence or reverse complementary sequence of the nucleotide sequence of the ribozyme recognition site I. The intron fragment IV is ligated to the 3′ end of truncated fragment III of the coding element, corresponding to the intron fragment IV, the second predetermined number of nucleotides at the 3′ end of truncated fragment III of the coding element constitute the ribozyme recognition site IV, and the sequence of the ribozyme recognition site IV is a reverse sequence or reverse complementary sequence of the nucleotide sequence of the ribozyme recognition site II.

In preparation of a circular RNA in vitro by the recombinant nucleic acid molecule containing the above elements, cleavage successively occurs at the ribozyme recognition site III and the ribozyme recognition site IV, releasing the intron fragment III and the intron fragment IV, and the 3′ end of the truncated fragment III of coding element is ligated to the 5′ end of the truncated fragment IV of the coding element to form the circular RNA molecule. As the ribozyme recognition site III and the ribozyme recognition site IV are provided inside the truncated fragments of the coding element, no additional E1 and E2 sequences are introduced into the circular RNA after in vitro circularization, providing advantages of accurate sequence, simple structure, low immunogenicity, and the like, enabling large-scale in vitro production with the application advantages in the fields of nucleic acid vaccines, expression of therapeutic proteins, clinical immunotherapy, and the like.

Translation Initiation Element

In the present disclosure, a translation initiation element may be any type of element that is able to initiate translation of a target polypeptide. In some embodiments, the translation initiation element is an element containing any one or more of the following sequences: an IRES sequence, a 5′UTR sequence, a Kozak sequence, a sequence with m⁶A modification (N (6) methyladenosine modification), and a complementary sequence of ribosome 18S rRNA. In other embodiments, the translation initiation element may be any other type of cap-independent translation elements.

In some embodiments, the translation initiation element is an IRES element derived from those including, but not limited to, viruses, mammals, fruit flies, and the like. In some alternative embodiments, the IRES element is derived from a virus. Illustratively, the IRES element contains an IRES sequence derived from a small RNA virus. Furthermore, the IRES element includes, but is not limited to, an IRES sequence derived from Echovirus, Human poliovirus, Human Enterovirus, Coxsackievirus, Human rhinovirus, Canine picornavirus, Turdivirus 3, Hepatovirus, Passerivirus, Picornaviridae, Tremovirus A, Feline kobuvirus, Murine kobuvirus, Kobuvirus sewage Kathmandu, Ferret kobuvirus, Marmot kobuvirus, Human parechovirus, Chicken picornavirus, Falcon picornavirus, Feline picornavirus, French Guiana picornavirus, and the like.

In some alternative embodiments, the recombinant nucleic acid molecule provided by the present disclosure consists of elements arranged in the following order in the 5′ to 3′ direction: an intron fragment II, a truncated fragment II of a coding element, a translation initiation element, a truncated fragment I of a coding element, and an intron fragment I. In other alternative embodiments, the recombinant nucleic acid molecule may also include any other one or more of the above elements, such as a transcriptional regulatory element for regulating transcription level, a translational regulatory element for regulating translation level, a purification element for purifying circular RNA, and the like.

In some alternative embodiments, the recombinant nucleic acid molecule provided by the present disclosure consists of elements arranged in the following order in the 5′ to 3′ direction: an intron fragment III, a truncated fragment IV of a coding element, a translation initiation element, a truncated fragment III of a coding element, and an intron fragment IV. In other alternative embodiments, the recombinant nucleic acid molecule may also include any other one or more of the above elements, such as a transcriptional regulatory element for regulating transcription level, a translational regulatory element for regulating translation level, a purification element for purifying circular RNA, and the like.

Intron Fragment

An intron fragment in the present disclosure is derived from Group I intron with ribozyme activity of self-splicing reaction and is widely found in various species. Illustratively, Group I introns include, but are not limited to, phage T4 td gene, Anabaena tRNA^(Leu), TpaCOX2, and Ptu, and the like.

In some embodiments, an intron fragment I and an intron fragment II are derived from the Group I intron, and contain a partial sequence close to the 5′ direction and another partial sequence close to the 3′ direction, respectively, both of which constitute the Group I intron. A ribozyme recognition site I is derived from an exon sequence (Exon 1, E1) ligated to the 5′ end of the Group I intron, and a ribozyme recognition site II is derived from an exon sequence (Exon 2, E2) ligated to the 3′ end of Group I intron. The intron fragment I is ligated to the ribozyme recognition site I, and the intron fragment II is ligated to the ribozyme recognition site II, thus forming a PIE system capable of self-splicing.

In other embodiments, an intron fragment III and an intron fragment IV are derived from the Group I intron, and contain a reverse sequence of the partial sequence close to the 5′ direction and a reverse sequence of another partial sequence close to the 3′ direction, respectively, both of which constitute the Group I intron. A ribozyme recognition site III is derived from a reverse sequence of the exon sequence (Exon 1, E1) ligated to the 5′ end of the Group I intron, and a ribozyme recognition site IV is derived from a reverse sequence of the exon sequence (Exon 2, E2) ligated to the 3′ end of the Group I intron. The intron fragment III is ligated to the ribozyme recognition site III, and the intron fragment IV is ligated to the ribozyme recognition site IV, thus forming a PIE system capable of self-splicing.

In some alternative embodiments, the ribozyme recognition site I consists of 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to 10 nucleotides. That is, the first predetermined number of nucleotides located at the 3′ end of truncated fragment I of the coding element is 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to nucleotides. Illustratively, the first predetermined number is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, and any integer value between any two of them.

In some alternative embodiments, the ribozyme recognition site II consists of 1 to 100 nucleotides, preferably 1 to 50 nucleotides, and more preferably 1 to 10 nucleotides. That is, the second predetermined number of nucleotides located at the 5′ end of truncated fragment II of the coding element is 1 to 100 nucleotides, preferably 1 to 50 nucleotides, and more preferably 1 to 10 nucleotides. Illustratively, the first predetermined number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and any integer value between any two of them.

In some alternative embodiments, the ribozyme recognition site III consists of 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to 10 nucleotides. That is, the first predetermined number of nucleotides located at the 5′ end of truncated fragment IV of the coding element is 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to 10 nucleotides. Illustratively, the first predetermined number is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, and any integer value between any two of them.

In some alternative embodiments, the ribozyme recognition site IV consists of 1 to 100 nucleotides, preferably 1 to 50 nucleotides, more preferably 1 to 10 nucleotides. That is, the second predetermined number of nucleotides located at the 3′ end of truncated fragment III of coding element is 1 to 100 nucleotides, preferably 1 to 50 nucleotides, and more preferably 1 to nucleotides. Illustratively, the first predetermined number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and any integer value between any two of them.

In some preferred embodiments, a sum of the first predetermined number and the second predetermined number is not equal to 3y, where y≥1 and y is an integer. That is, the sum of the first predetermined number and the second predetermined number is not equal to an integer multiple of 3. When the sum is not a value of an integer multiple of 3, increased freedom of arranging a ribozyme recognition site in the coding fragment may be obtained, resulting in effective circularization for the circular RNA.

In some alternative embodiments, the Group I intron is a T4 td intron derived from a T4 phage td gene, with a secondary structure shown in FIG. 13 . The nucleotide sequence of the ribozyme recognition site used for circularization in the T4 td intron is “5′-TTGGGTCT-3′”, where the circularization position is located between T and C. Therefore, the nucleotide sequence of the ribozyme recognition site I is “5′-TTGGGT-3′”, and the nucleotide sequence of the ribozyme recognition site II is “5′-CT-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-TGGGTT-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-TC-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-ACCCAA-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-AG-3′”.

It should be noted that the ribozyme recognition site with a few base mutations may also be used for in vitro circularization of a circular RNA, under conditions that keep the bases at the circularization position unchanged. Illustratively, the present disclosure has found that the ribozyme recognition site and ligated intron fragments thereof may remain the circularization activity, in the presence of one or more of the following mutations in “5′-TTGGGTCT-3”: base T to C mutation at position 2, base G to A mutation at position 3, and base T to A mutation at position 8.

In some alternative embodiments, the nucleotide sequence of the intron fragment I derived from the T4 td intron is shown in SEQ ID NO: 7, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to that shown in SEQ ID NO: 7.

In some alternative embodiments, the nucleotide sequence of the intron fragment II derived from the T4 td intron is shown in SEQ ID NO: 6, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to that shown in SEQ ID NO: 6.

In some alternative embodiments, the Group I intron is a TpaCOX2 intron, which is an intron sequence of a cytochrome xoidase cox2 gene of a T. papilionaceus mitochondria, with a secondary structure as shown in FIG. 14 . The nucleotide sequence of the ribozyme recognition site used for circularization in the TpaCOX2 intron is “5′-ACGTCTTAACCAA-3′” (SEQ ID NO: 80), where the circularization position is located between T and C. Therefore, the nucleotide sequence of the ribozyme recognition site I is “5′-ACGTCTT-3′”, and the nucleotide sequence of the ribozyme recognition site II is “5′-AACCAA-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-TTCTGCA-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-AACCAA-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-AAGACGT-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-TTGGTT-3′”.

In some alternative embodiments, the Group I intron is a Ptu intron, with a secondary structure shown in FIG. 15 . The Ptu is a precursor RNA of a chloroplast ribosomal large subunit RNA (rrnL) in Pedinomonas tuberculata, which is a green algae in Pseudomonas. The nucleotide sequence of the ribozyme recognition site used for circularization in the Ptu intron is “5′-AGGGATCA-3′”, where the circularization position is located between T and C. Therefore, the nucleotide sequence of the ribozyme recognition site I is “5′-AGGGAT-3′”, and the nucleotide sequence of the ribozyme recognition site II is “5′-CA-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-TAGGGA-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-AC-3′”. Alternatively, the nucleotide sequence of the ribozyme recognition site III is “5′-ATCCCT-3′”, and the nucleotide sequence of the ribozyme recognition site IV is “5′-TG-3′”.

It should be noted that the sequences of the ribozyme recognition sites and the intron fragments are not limited in the present disclosure, as long as they are derived from the Group I introns, efficient for circularization, and may be used to prepare a circular RNA in vitro.

Recombinant Nucleic Acid Molecule Containing Insertion Element

In some embodiments, a recombinant nucleic acid molecule includes an insertion element, which may be used to regulate transcription of the recombinant nucleic acid molecule, regulate translation of a circular RNA, enable specific expression of a circular RNA between different tissues, or purify a circular RNA, and the like. Illustratively, as shown FIG. 2B, the insertion element is located between a truncated fragment II of a coding element and a translation initiation element. Alternatively, as shown in FIG. 2E, the insertion element is located between a truncated fragment IV of a coding element and a translation initiation element. Specifically, the insertion element is ligated to the 5′ end of the translation initiation element.

In some embodiments, the insertion element is at least one selected from the group consisting of (i) to (iii): (i) a transcriptional regulatory element, (ii) a translational regulatory element, and (iii) a purification element. Illustratively, the insertion element includes one of or a combination of any two or more of the following sequences: an untranslated region (UTR) sequence, a polyN sequence, an aptamer sequence, a riboswitch sequence, and a transcriptional regulator-binding sequence. In the polyN sequence, N is selected from at least one of A, T, G, and C.

In some alternative embodiments, the translational regulatory element includes an untranslated region sequence, which may serve to regulate stability, immunogenicity, and efficiency of the circular RNA in expressing the target polypeptide, and the like. The untranslated region sequence is not specifically limited in the present disclosure, which may be selected from any type of sequences known in the art having the properties of regulating the circular RNA transcription, translation, intracellular stability, immunogenicity, and the like. Furthermore, the untranslated region sequence is also not limited to the 5′UTR sequence or the 3′UTR sequence.

In some alternative embodiments, the untranslated region sequence contains one or more (for example, 1, 2, 3, 4, 5, 6, and 7) miRNA recognition sequences. The addition of one or more miRNA recognition sequences may provide specific expression of a circular RNA in different tissues and cells, providing targeted delivery of a circular RNA molecule.

In some alternative embodiments, the translational regulatory element contains a polyN sequence, where N may be at least one of A, T, G, and C. The addition of the translation regulatory element containing the polyN sequence may improve efficiency of target polypeptide expression of the circular RNA, immunogenicity, and stability of the circular RNA, or purify the circular RNA. The length of polyN sequence, as well as the selected N type and the manner of composition in the polyN sequence, is not specifically limited in the present disclosure, as long as they facilitate the improvement of the properties of the circular RNA. Illustratively, polyN sequences are polyA sequence, polyAC sequence, and the like.

In some alternative embodiments, the translational regulatory element contains a riboswitch sequence. The riboswitch sequence is a class of untranslated sequences capable of regulating the transcription and translation of RNA. The riboswitch sequence in the present disclosure may affect expression of the circular RNA, including but not limited to transcription termination, inhibition of translation initiation, mRNA self-cleavage, and changes of splicing pathway in eukaryotes. In addition, the riboswitch sequence may also control the expression of circular RNA by triggering binding or removing of molecules. Illustratively, riboswitch sequences are cobalamin riboswitch (also called B₁₂-element), FMN riboswitch (also called RFN element), glmS riboswitch, SAM riboswitch, SAH riboswitch, tetrahydrofolate riboswitch, Moco riboswitch, and the like, and the type and sequence of riboswitch sequence are not limited in the present disclosure, as long as they can realize regulation of the transcriptional and translational levels of circular RNAs expressing target polypeptides.

In some alternative embodiments, the translational regulatory element contains an aptamer sequence. In the present disclosure, the aptamer sequence may be used to regulate transcription and translation of circular RNAs, or to purify and prepare a circular RNA in vitro.

In an exemplary embodiment, the aptamer sequence is the sequence as shown in SEQ ID NO: 37, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to that shown in SEQ ID NO: 37.

Recombinant Nucleic Acid Molecule Containing Homology Arm

In some embodiments, a recombinant nucleic acid molecule contains a homology arm. Specifically, the homology arm includes a 5′ homology arm located at the 5′ end of the recombinant nucleic acid molecule and a 3′ homology arm located at the 3′ end of the recombinant nucleic acid molecule, and the nucleic acid sequence of the 5′ homology arm hybridizes with the nucleotide sequence of the 3′ homology arm.

In some embodiments, in the recombinant nucleic acid molecule, the 5′ homology arm is ligated to the 5′ end of an intron fragment II, and the 3′ homology arm is ligated to the 3′ end of an intron fragment I, as shown in FIG. 2C. The hybridization between the sequence of 5′ homology arm with that of 3′ homology arm, allows intron fragment I and intron fragment II to be close to each other, and after the cleavage at a linking position between a ribozyme recognition site I and the intron fragment I, it is favorable for 3′-OH at ribozyme recognition site I to further attack a phosphodiester bond linking ribozyme recognition site II and intron fragment II, thereby releasing intron fragment II.

In some embodiments, in the recombinant nucleic acid molecule, the 5′ homology arm is ligated to the 5′ end of the intron fragment III, and the 3′ homology arm is ligated to the 3′ end of the intron fragment IV, as shown in FIG. 2F. The hybridization between the sequence of 5′ homology arm with that of 3′ homology arm, allows the intron fragment III and the intron fragment IV to be close to each other, after the cleavage at a linking position between a ribozyme recognition site III and the intron fragment III, it is favorable for 3′-OH at ribozyme recognition site III to further attack a phosphodiester bond linking ribozyme recognition site IV and intron fragment IV, thereby releasing intron fragment IV.

In some embodiments of the present disclosure, by comparing the effects on in vitro circularization of the circular RNA when the homology arm is added or not added. it is discovered in the present disclosure that effective circularization rate may still be remained for the recombinant nucleic acid molecule without the homology arm added, as compared to recombinant nucleic acid molecule with the homology arm added. Therefore, in order to further simplify structure of the recombinant nucleic acid molecule, in some embodiments, the 5′ homology arm and the 3′ homology arm are not contained in the recombinant nucleic acid molecule. This is because within the frame of the present disclosure, the circularization site (ribozyme recognition site) always separates the coding gene into two parts, for which typically no complicated secondary structure exists in the coding region. Such benign sequence separates a promoter element from a self-splicing intron sequence, thus forming a unique secondary structure, which is more conducive to correct folding and approaching of the intron sequences. As a result, an effective circularization may be achieved without the homology arm within the frame of the present disclosure.

Target Polypeptide

The type of target polypeptide is not limited in the present disclosure, which may be a human-derived protein or a non-human protein. Illustratively, a target polypeptide include, but are not limited to, an antigen, an antibody, an antigen-binding fragment, a fluorescent protein, a protein with therapeutic activity against diseases, and a protein with gene-editing activity.

The term “antibody”, as used herein in the broadest sense thereof, refers to a protein containing an antigen-binding site, which encompasses natural antibodies and artificial antibodies with various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi specific antibodies (for example, bispecific antibodies), single-stranded antibodies, complete antibodies, and antibody fragments.

The term “antigen-binding fragment” herein is a portion or a fragment of a intact or complete antibody with fewer amino acid residues than the intact or complete antibody, and is is capable of binding to an antigen or competing with the intact antibody (that is, the intact antibody from which the antigen-binding fragment is derived) for binding the antigen. An antigen-binding fragment of an antibody may be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. The antigen-binding fragment includes, but is not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂, a diabody, a linear antibody, a single chain antibody (e.g., ScFv), a single domain antibody, a bivalent or bispecific antibody or a fragment thereof; Camelidae antibody (a heavy chain antibody); and a bispecific antibody or polyspecific antibody formed by antibody fragments.

In the present disclosure, a protein with therapeutic activity against diseases may include, but is not limited to, an enzyme replacement protein, a protein for supplementation, a protein vaccine, an antigen (e.g. a tumor antigen, a virus, bacteria), a hormone, a cytokine, an antibody, an immunotherapy (e.g., a cancer), a cell reprogramming/transdifferentiation factor, a transcription factor, a chimeric antigen receptor, a transposase or nuclease, an immune effector (e.g., having effects on susceptibility to immune response/signal), a regulated death effector protein (e.g., an inducer of apoptosis or necrosis), an insoluble inhibitor of a tumor (e.g., a cancer protein inhibitor), an epigenetic modifier, an epigenetic enzyme, a transcription factor, a DNA or protein-modifying enzyme, a DNA intercalator, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, an enzyme competition inhibitor, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand or receptor, and the CRISPR system or a component thereof, and the like.

Coding Element for Forming One or More Coding Regions

In some embodiments, the recombinant nucleic acid molecule is used for preparing a circular RNA containing a coding element, where the coding element in the circular RNA includes a coding region 1, optionally (a) at least one coding region 2, and optionally (b) at least one coding region 3.

In the present disclosure, a truncated fragment I of the coding element and a truncated fragment II of the coding element in the recombinant nucleic acid molecule form the coding region 1, optionally (a) the at least one coding region 2, and optionally (b) the at least one coding region 3. Illustratively, in some embodiments, the truncated fragment I of the coding element and the truncated fragment II of the coding element form the coding region 1, as shown in FIG. 3A. Accordingly, the truncated fragment I of the coding element is a truncated fragment I of the coding element 1, the truncated fragment II of the coding element is a truncated fragment II of the coding region 1. Therefore, the 3′ end of truncated fragment I of the coding region 1 contains a ribozyme recognition site I that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment I of the coding region 1; and the 5′ end of the truncated fragment II of the coding region 1 contains a ribozyme recognition site II that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment II of coding region 1. The truncated fragment I of the coding element and the truncated fragment II of the coding element in the recombinant nucleic acid molecule are used to form the coding region 1, to express a target polypeptide in vitro or in vivo.

In other embodiments, the truncated fragment I of the coding element and the truncated fragment II of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1 and at least one coding region 2. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least two target polypeptides. Illustratively, the recombinant nucleic acid molecule contains elements arranged in the following order: (i) an intron fragment II, a truncated fragment II of the coding region 1, the at least one coding region 2, a translation initiation element, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 4A to 4B.

In some alternative embodiments, the coding regions 2 has a quantity of one, and the recombinant nucleic acid molecule contains elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, the coding region 2, a translation initiation element, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 4A. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding regions 2 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, the at least two coding regions 2, a translation initiation element, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 4B. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment II of the coding region 1 and the coding region 2, and/or a linker located between any two adjacent coding regions 2. Such linkers are used to separate the coding region 2 from the coding region 1, and separate any two adjacent coding regions 2 from each other, allowing the circular RNA prepared by the recombinant nucleic acid molecule to express two or more target polypeptides.

In other embodiments, the truncated fragment I of the coding element and the truncated fragment II of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1 and at least one coding region 3. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least two target polypeptides. Illustratively, the recombinant nucleic acid molecule includes elements arranged in the following order: (ii) an intron fragment II, a truncated fragment II of the coding region 1, a translation initiation element, the at least one coding region 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 5A to 5B.

In some alternative embodiments, the coding regions 3 has a quantity of one, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, a translation initiation element, the coding region 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 5A. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding regions 3 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, a translation initiation element, the at least two coding regions 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 5B. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment I of the coding region 1 and the coding region 3, and/or a linker located between any two adjacent coding regions 3. Such linkers are used to separate the coding region 3 from the coding region 1, and separate any two adjacent coding regions 3 from each other, allowing the circular RNA prepared by the recombinant nucleic acid molecule to express two or more target polypeptides.

In other embodiments, the truncated fragment I of the coding element and the truncated fragment II of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1, at least one coding region 2, and at least one coding region 3. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least three target polypeptides. Illustratively, the recombinant nucleic acid molecule includes elements arranged in the following order: (iii) an intron fragment II, a truncated fragment II of the coding region 1, the at least one coding region 2, a translation initiation element, the at least one coding region 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIGS. 6A to 6B.

In some alternative embodiments, the coding region 2 and the coding region 3 has a quantity of one respectively, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, the coding region 2, a translation initiation element, the coding region 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 6A. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 2 has a quantity of at least two, and the coding region 3 has a quantity of at least two. The recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, a truncated fragment II of the coding region 1, the at least two coding regions 2, a translation initiation element, the at least two coding regions 3, a truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 6B. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment II of the coding region 1 and the coding region 2, a linker located between the truncated fragment I of the coding region 1 and the coding region 3, a linker located between any two adjacent coding regions 2, and/or a linker located between any two adjacent coding regions 3. Such linkers are used to separate the coding region 2 from the coding region 1, the coding region 3 from the coding region 1, any two adjacent coding regions 2 from each other, and any two adjacent coding regions 3 from each other, allowing the circular RNA prepared by the recombinant nucleic acid molecule to express three or more target polypeptides.

In some embodiments, a truncated fragment III of the coding element and a truncated fragment IV of the coding element are used to form a coding region 1. Accordingly, the truncated fragment III of the coding element is the truncated fragment III of the coding region 1, and the truncated fragment IV of the coding element is the truncated fragment IV of the coding region 1, as shown in FIG. 3B. Therefore, the 3′ end of truncated fragment III of the coding region 1 contains a ribozyme recognition site IV that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment III of the coding region 1; and the 5′ end of truncated fragment IV of the coding region 1 contains a ribozyme recognition site III that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment IV of coding region 1. The truncated fragment III of the coding element and the truncated fragment IV of the coding element in the recombinant nucleic acid molecule are used to form the coding region 1, to express a target polypeptide in vitro or in vivo.

In other embodiments, the truncated fragment III of the coding element and the truncated fragment IV of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1 and at least one coding region 2. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least two target polypeptides. Illustratively, the recombinant nucleic acid molecule includes elements arranged in the following order: (v) an intron fragment III, the truncated fragment IV of the coding region 1, the at least one coding region 2, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 4C to 4D.

In some alternative embodiments, the coding fragment 2 has a quantity of one, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, the coding region 2, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 4C. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 2 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, the at least two coding regions 2, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 4D. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment IV of the coding region 1, and the coding region 2, and/or a linker located between any two adjacent coding regions 2. Such linkers are used to separate the coding region 2 from the coding region 1, and any two adjacent coding regions 2 from each other, allowing the circular RNA prepared by the recombinant nucleic acid molecule to express two or more target polypeptides.

In other embodiments, the truncated fragment III of the coding element and the truncated fragment IV of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1 and at least one coding region 3. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least two target polypeptides. Illustratively, the recombinant nucleic acid molecule includes elements arranged in the following order: (vi) an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the at least one coding region 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 5C to 5D.

In some alternative embodiments, the coding region 3 has a quantity of one, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the coding region 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 5C. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 3 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the at least two coding regions 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 5D. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment III of the coding region 1 and the coding region 3, and/or a linker located between any two adjacent coding regions 3. Such linkers are used to separate the coding region 3 from the coding region 1, and any two adjacent coding regions 3 from each other, allowing the circular RNA prepared by the recombinant nucleic acid molecule to express two or more target polypeptides.

In other embodiments, the truncated fragment III of the coding element and the truncated fragment IV of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1, at least one coding region 2, and at least one coding region 3. The circular RNA prepared by the recombinant nucleic acid molecule may provide tandem expression of at least three target polypeptides. Illustratively, the recombinant nucleic acid molecule includes elements arranged in the following order: (vii) an intron fragment III, the truncated fragment IV of the coding region 1, the at least one coding region 2, a translation initiation element, the at least one coding region 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 6C to 6D.

In some alternative embodiments, the coding region 2 has a quantity of one, and the coding region 3 has a quantity of one. The recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, the coding region 2, a translation initiation element, the coding region 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 6C. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding regions 2 has a quantity of at least two, and the coding region 3 has a quantity of at least two. The recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, the at least two coding regions 2, a translation initiation element, the at least two coding regions 3, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 6D. In other alternative embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some preferred embodiments, the recombinant nucleic acid molecule further includes a linker located between the truncated fragment IV of the coding region 1, and the coding region 2, a linker located between the truncated fragment III of the coding region 1 and the coding region 3, a linker located between any two adjacent coding regions 2, and/or a linker located between any two adjacent coding regions 3. Such linkers are used to separate the coding region 2 from the coding region 1, the coding region 3 from the coding region 1, any two adjacent coding regions 2 from each other, and any two adjacent coding regions 3 from each other, enabling the circular RNA prepared by the recombinant nucleic acid molecule to express two or more target polypeptides.

In the present disclosure, the linker may be a polynucleotide encoding 2A peptide, or another type of polynucleotide encoding a linker peptide for spacing target polypeptides, where the 2A peptide is a short peptide (about 18 to 25 amino acids in length) derived from a virus, commonly known as a “self-splicing” peptide, enabling a strand of transcript to produce a variety of proteins. Illustratively, 2A peptides are P2A, T2A, E2A, F2A, and the like.

In the present disclosure, the coding region 1, each coding region 2 and each coding region 3 encode any type of target polypeptide independently of one another. The target polypeptides encoded by the coding region 1 and any one coding region 2 may be the same or different, the target polypeptide encoded by the coding region 1 and any one coding region 3 may be the same or different, the target polypeptide encoded by any two coding regions 2 may be the same or different, the target polypeptide encoded by any two coding regions 3 may be the same or different, and the target polypeptide encoded by any one coding region 2 and any one coding region 3 may be the same or different.

In the present disclosure, the coding regions located at different positions are distinguished by numbering the coding regions (for example, coding region 1, coding region 2, coding region 3, and the like). Illustratively, the coding region 1 represents a coding region containing a ribozyme recognition site; the coding region 2 represents a coding region between the truncated fragment II of the coding region 1 and the translation initiation element, or the coding region 2 represents a coding region between the truncated fragment IV of the coding region 1 and the translation initiation element; the code region 3 represents a coding region between the truncated fragment I of the coding region 1 and the translation initiation element, or the coding region 2 represents a coding region between the truncated fragment III of the coding region 1 and the translation initiation element.

Recombinant Nucleic Acid Molecule Including at Least Two Translation Initiation Elements

In some embodiments, the recombinant nucleic acid molecule is used for preparing a circular RNA containing a coding element; where the coding element in the circular RNA contains a coding region 1, at least one coding region 4, and a translation initiation element located between any two adjacent coding regions.

In some embodiments, a truncated fragment I of the coding element and a truncated fragment II of the coding element in the recombinant nucleic acid molecule form the coding region 1, the at least one coding region 4, and the translation initiation element located between any two adjacent coding regions. The 3′ end of the truncated fragment I of the coding element contains a ribozyme recognition site I that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment I of the coding region 1; and the 5′ end of the truncated fragment II of the coding element contains a ribozyme recognition site II that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment II of the coding region 1. In the circular RNA prepared by the recombinant nucleic acid molecule in vitro, each coding region corresponds to one translation initiation element, allowing for tandem expression of at least two target polypeptides.

In some alternative embodiments, the coding region 4 has a quantity of one, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, the truncated fragment II of the coding region 1, a translation initiation element, the coding region 4, a translation initiation element, the truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 7A. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 4 has a quantity of two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, the truncated fragment II of the coding region 1, a translation initiation element, the coding region 4, a translation initiation element, the coding region 4, a translation initiation element, the truncated fragment I of the coding region 1, and an intron fragment I, as shown in FIG. 7B. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, as shown in FIG. 7C, the coding region 4 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment II, the truncated fragment II of the coding region 1, a translation initiation element, the at least one coding region 4, a translation initiation element, the at least one coding region 4, a translation initiation element, the truncated fragment I of the coding region 1, and an intron fragment I; where one translation initiation element is contained between any two coding regions 4. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some other embodiments, the truncated fragment III of the coding element and the truncated fragment IV of the coding element in the recombinant nucleic acid molecule are used to form a coding region 1, at least one coding region 4, and a translation initiation element located between any two adjacent coding regions. The 3′ end of the truncated fragment III of the coding region 1 contains a ribozyme recognition site IV that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment III of the coding region 1; and the 5′ end of the truncated fragment IV of the coding region 1 contains a ribozyme recognition site III that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment IV of the coding region 1. In the circular RNA prepared by the recombinant nucleic acid molecule in vitro, each coding region corresponds to one translation initiation element, allowing for tandem expression of at least two target polypeptides.

In some alternative embodiments, the coding region 4 has a quantity of one, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the coding region 4, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 7D. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 4 has a quantity of two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the coding region 4, a translation initiation element, the coding region 4, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV, as shown in FIG. 7E. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In some alternative embodiments, as shown in FIG. 7F, the coding region 4 has a quantity of at least two, and the recombinant nucleic acid molecule includes elements arranged in the following order: an intron fragment III, the truncated fragment IV of the coding region 1, a translation initiation element, the at least one coding region 4, a translation initiation element, the at least one coding region 4, a translation initiation element, the truncated fragment III of the coding region 1, and an intron fragment IV; where one translation initiation element is contained between any two coding regions 4. In other embodiments, the recombinant nucleic acid molecule consists of the elements arranged in the above order.

In the present disclosure, the coding region 1 and each coding region 4 encode any type of target polypeptide independently of one another. The target polypeptides encoded by the coding region 1 and any one coding region 4 may be the same or different, and the target polypeptides encoded by any coding region 4 may be the same or different.

In the circular RNA prepared by the recombinant nucleic acid molecule above, the 5′ end of each coding region is correspondingly ligated to one translation initiation element, and the coding region 1 and the at least one coding region 4 are ligated in tandem by a plurality of translation initiation elements, allowing for expression of at least two target polypeptides.

In the present disclosure, the coding regions located at different positions are distinguished by numbering the coding regions (for example, coding region 1, coding region 4, etc.). Illustratively, the coding region 1 represents a coding region containing a ribozyme recognition site; the code region 4 represents a coding region between the truncated fragment I of the coding region 1 and the truncated fragment II of the coding region 2, or the coding region 4 represents a coding region between the truncated fragment III of the coding region 1 and the truncated fragment IV of the coding region 2.

Recombinant Expression Vector Containing Recombinant Nucleic Acid Molecule

In some embodiments, the recombinant nucleic acid molecule serves as a portion of a recombinant expression vector for preparing a circular RNA. The circular RNAs expressing a target polypeptide may be prepared in a process of transcription and circularization in vitro.

In other embodiments, the recombinant nucleic acid molecule may serve as a circularization precursor RNA molecule, or a part thereof obtained after linearization and transcription of the recombinant expression vector. That is, only circularization of the recombinant nucleic acid molecule is needed to obtain the circular RNA expressing a target polypeptide.

In some embodiments, the steps of preparing a circular RNA in vitro include:

-   -   transcribing the recombinant nucleic acid molecule according to         any one item described above or the recombinant expression         vector described above to form a circularization precursor         nucleic acid molecule; and     -   circularizing the circularization precursor nucleic acid to         obtain the circular RNA.

In some alternative embodiments, the method further includes a step of purifying the circular RNA.

Circular RNA

In some embodiments, the circular RNA of the present disclosure is prepared by using a Clean PIE system provided by the present disclosure, and includes elements arranged in the following order in the 5′ to 3′ direction: a translation initiation element, and a coding element for encoding at least one target polypeptide.

Compared with the circular RNA prepared by a classic PIE system shown in FIG. 8B, the circular RNA prepared by the Clean PIE system of the present disclosure does not introduce additional E1 and E2 sequences (FIG. 8A) under conditions ensuring integrity of protein-coding sequences, to guarantee sequence accuracy and secondary structure of the circular RNA, reduce natural immunogenicity of the circular RNA, and improve stability thereof in cells, thus making it suitable for clinical application fields, such as serving as a gene therapy vector, an expressing therapeutic protein, serving as a nucleic acid vaccine and the like, showing broad application prospects.

In some embodiments, the coding element of the circular RNA includes a coding region 1 and at least one in the group consisting of (i) to (ii): (i) at least one coding region 2, and (ii) at least one coding region 3. Each coding region encodes any type of target polypeptide independently of one another, and the circular RNA may encode one or more target polypeptides in tandem. Illustratively, the circular RNA expresses 1, 2, 3, 4, 5, 10, 15, 20, 25, or another number of target polypeptides.

As a preferred embodiment, two adjacent coding regions are linkeded by a linker, which is used to encode a linker peptide to separate target polypeptides encoded by the adjacent coding regions, enabling the same circular RNA to encode two or more target polypeptides.

In some alternative embodiments, the circular RNA contains a translation initiation element, a coding region 1, a coding region 2, and a linker located between the coding region 1 and the coding region 2 in the 5′ to 3′ direction, as shown in FIG. 9A. The linker separates the coding region 1 from the coding region 2, enabling the circular RNA to express at least two target polypeptides in tandem in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 2 may have a quantity of two or more. Illustratively, the circular RNA contains a translation initiation element, a coding region 1, two coding regions 2 (for convenience of description, designated as a first coding region 2 and a second coding region 2 in the 5′ to 3′ direction), a linker located between the coding region 1 and the first coding region 2, and a linker located between the first coding region 2 and the second coding region 2 in the 5′ to 3′ direction, as shown in FIG. 10A. The above-mentioned circular RNA may allow for tandem expression of three or more target polypeptides in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In some alternative embodiments, the circular RNA contains a translation initiation element, a coding region 3, a coding region 1, and a linker located between the coding region 1 and the coding region 3 in the 5′ to 3′ direction, as shown in FIG. 9B. The linker separates the coding region 1 from the coding region 3, enabling the circular RNA to express at least two target polypeptides in tandem in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 3 may have a quantity of two or more. Illustratively, the circular RNA contains a translation initiation element, two coding regions 3 (for convenience of description, designated as a first coding region 3 and a second coding region 3 in the 5′ to 3′ direction), a coding region 1, a linker located between the coding region 1 and the second coding region 3, and a linker located between the first coding region 3 and the second coding region 3 in the 5′ to 3′ direction, as shown in FIG. 10C. The above-mentioned circular RNA may allow for tandem expression of three or more target polypeptides in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In some alternative embodiments, the circular RNA contains both a coding region 2 and a coding region 3. Illustratively, the circular RNA contains a translation initiation element, a coding regions 3, a coding region 1, a coding region 2, a linker located between the coding region 1 and the coding region 3, and a linker located between coding region 1 and coding region 2 in the 5′ to 3′ direction, as shown in FIG. 10B. The above-mentioned circular RNA may allow for tandem expression of three or more target polypeptides in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In the present disclosure, the coding region 1, each coding region 2 and each coding region 3 encode any type of target polypeptide independently of one another. The target polypeptide encoded by the coding region 1 and any one coding region 2 may be the same or different, the target polypeptide encoded by the coding region 1 and any one coding region 3 may be the same or different, the target polypeptide encoded by any two coding regions 2 may be the same or different, the target polypeptide encoded by any two coding regions 3 may be the same or different, and the target polypeptide encoded by any one coding region 2 and any one coding region 3 may be the same or different.

In some embodiments, the coding regions of the circular RNA includes one coding region 1 and at least one coding region 4, and the 5′ end of any one coding region is ligated to a translation initiation element. Illustratively, the coding region 4 has a quantity of 1, 2, 3, 4, 5, 10, 15, 20, 25, and the like. The transcription of different coding regions is initiated by using a translation initiation element ligated to the 5′ end of each coding region, enabling the same circular RNA to encode two or more target polypeptides.

In some alternative embodiments, the coding region 4 has a quantity of one, and the circular RNA contains a translation initiation element, a coding region 1, a translation initiation element, and a coding region 4 in the 5′ to 3′ direction, as shown in FIG. 11A. The above-mentioned circular RNA may allow for tandem expression of two target polypeptides in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In some alternative embodiments, the coding region 4 may have a quantity of at least two. Illustratively, the circular RNA contains a translation initiation element, a coding region 1, a translation initiation element, at least two coding regions 4, and a translation initiation element located between the at least two coding regions 4 in the 5′ to 3′ direction, as shown in FIG. 11B. The above-mentioned circular RNA may allow for tandem expression of two target polypeptides in cells. In other alternative embodiments, the circular RNA consists of the elements arranged in the above order.

In the present disclosure, the coding region 1 and each coding region 4 encode any type of target polypeptide independently of one another. The target polypeptide encoded by the coding region 1 and any one coding region 4 may be the same or different, and the target polypeptide encoded by any two coding regions 4 may be the same or different.

In some alternative embodiments, the circular RNA contains an insertion element. The insertion element is ligated to the 5′ end of any translation initiation element.

Methods for Screening Target Coding Region Sequence Containing Ribozyme Recognition Site

In some embodiments, the present disclosure provides a method for screening a target coding region sequence containing a ribozyme recognition site, including the following steps:

S1, extracting m amino acid units from the target polypeptide containing q amino acids in the N-terminal-to-C-terminal direction, with each of amino acid units containing n amino acids; where at least one repeated amino acid is contained between any two adjacent amino acid units, n is an integer and n≥2, m is an integer and m≥1.

Specifically, in the N-terminal-to-C-terminal direction, the extracted m amino acid units are sequentially designated as amino acid units R₁ to R_(M). A quantity of the amino acids in each amino acid unit from R₁ to R_(M) is n, and at least one identical amino acid is contained between any two adjacent amino acid units, with a quantity of amino acid units is any integer from 1 to (n−1). As a preferred embodiment, the quantity of repeated amino acids between any two adjacent amino acid units is n−1.

Illustratively, the target polypeptide sequentially consists of AA₁ to AA_(q) amino acids in the N-terminal-to-C-terminal direction. When extracting the amino acids from the target polypeptide, a total number of n amino acids (AA₁ included) are extracted as an amino acid unit R₁, with AA₁ as a start amino acid.

Furthermore, when extracting the amino acid unit R₂, a start position for extracting the amino acid unit R₂ may be any position from positions [2] to [n] in the amino acid unit R₁. For example, when n is 2, a start amino acid of the amino acid unit R₂ is the amino acid at the position [2] in the amino acid unit R₁. When n is 3, the start amino acid of the amino acid unit R₂ is the amino acid at the position [2] or [3] in the amino acid unit R₁ (AA₂ or AA₃). When n is 4, the start amino acid of the amino acid unit R₂ is the amino acid at the position [2], [3] or [4] in the amino acid unit R₁ (AA₂, AA₃ or AA₄). Analogy may be made with the increase of n, which is not exhaustive herein.

As a preferred embodiment, the start amino acid of the amino acid unit R₂ is the amino acid at the position [2] in the amino acid unit R₁. For example, when n is any integer of 2 or more, a start amino acid of the amino acid unit R₂ is always AA₂.

Furthermore, when extracting the amino acid unit R₃, a start position for extracting the amino acid unit R₃ may be any position from position [2] to [n] in the amino acid unit R₂. For example, when n is 2, a start amino acid of the amino acid unit R₃ is the amino acid at the position [2] in the amino acid unit R₂. When n is 3, the start amino acid of the amino acid unit R₃ is the amino acid at the position [2] or [3] in the amino acid unit R₂. When n is 4, the start amino acid of the amino acid unit R₃ is the amino acid at the position [2], [3] or [4] in the amino acid unit R₂. Analogy may be made with the increase of n, which is not exhaustive herein.

As a preferred embodiment, the start amino acid of the amino acid unit R₃ is the amino acid at the position [2] in the amino acid unit R₂. For example, when n is any integer of 2 or more, the start amino acid of the amino acid unit R₃ is always AA₃.

Furthermore, the start position for extracting the amino acid unit R₄ may be any position from positions [2] to [n] in the amino acid unit R₃. For example, when n is 2, the start amino acid of the amino acid unit R₄ is the amino acid at a position [2] in the amino acid unit R₃. When n is 3, the start amino acid of the amino acid unit R₄ is the amino acid at the position [2] or [3] in the amino acid unit R₃. When n is 4, the start amino acid of the amino acid unit R₄ is the amino acid at the position [2], [3] or [4] in the amino acid unit R₃. Analogy may be made with the increase of n, which is not exhaustive herein.

As a preferred embodiment, the start amino acid of the amino acid unit R₄ is the amino acid at the position [2] in the amino acid unit R₃. For example, when n is any integer of 2 or more, the start amino acid of the amino acid unit R₄ is always AA₄.

The extraction of amino acid units is carried out in the above manner until the amino acid unit R_(m) is extracted.

In a preferred embodiment, a target polypeptide sequence consisting of q amino acids is split, with a step of 1 and a window length of n, to obtain m amino acid units, where m=q+1−n, n is an integer and n≥2, m is an integer and m≥1.

S2, determining m codon sequence sets, where each of the codon sequence sets includes a codon sequence corresponding to each of the amino acid unit.

Specifically, according to the degeneracy principle of amino acid codons, the codon sequence set corresponding to each amino acid unit is obtained. Codon sequence sets C₁-C_(m) are obtained corresponding to amino acid units R₁-R_(m).

S3, traversing the m codon sequence sets to obtain a matching value of each codon sequence in each of the codon sequence sets with a target motif.

Specifically, the target motif contains a ribozyme recognition site sequence that is formed by ligating the nucleotide sequence of a ribozyme recognition site I to the nucleotide sequence of a ribozyme recognition site II, or formed by ligating the nucleotide sequence of a ribozyme recognition site III to the nucleotide sequence of a ribozyme recognition site IV. Illustratively, ribozyme recognition site sequences include, but are not limited to, “5′-TTGGGTCT-3′”, “5′-ACGTCTTAACCAA-3′”, “5′-AGGGATCA-3′”, and the like.

Furthermore, the target motif also contains x nucleotide(s) ligated to at least one of the 5′ end and the 3′ end of the ribozyme recognition site sequence, to form a target motif consisting of 3n nucleotides. Each x is an integer≥0 independently of one another, and each ligated nucleotide is selected from any type of nucleotide independently of one another. For example, when the ribozyme recognition site sequence is “5′-TTGGGTCT-3′”, the target motif may correspond to at least one selected from the following (a1) to (a6), where X=A, T, C, G:

(a1) TTGGGTCTX; (a2) XTTGGGTCT; (a3) XTCTGGGTT; (a4) TCTGGGTTX; (a5) XXTTGGGTCTXX; (a6) XXTCTGGGTTXX.

In some embodiments, the step of traversing m codon sequence sets to obtain a matching value of each codon sequence in each of codon sequence sets with a target motif includes aligning each codon sequence in the codon sequence sets C₁-C_(m) with the target motif to calculate the matching value of each codon subsequence.

Furthermore, the target motif contains an effective base pair that corresponds to two bases at a linking position of the ribozyme recognition site I and the ribozyme recognition site II. That is, the effective base pair refers to the two bases used for circularization in the ribozyme recognition site. For example, when the sequence of ribozyme recognition site is “5′-TTGGGTCT-3”, the effective base pair refers to TC therein. When the sequence of the ribozyme recognition site is “5′-ACGTCTTAACCAA-3”, the effective base pair refers to TA therein. When the sequence of ribozyme recognition site is “5′-AGGGATCA-3”, the effective base pair refers to TC therein.

When aligning each codon sequence with the target motif, it is determined whether bases at positions in the codon sequence corresponding to the effective base pair are effective bases, and if the codon sequence does not contain the effective base pair, then an alignment value of the codon sequence is not outputted.

If the codon sequence contains the effective base pair, the alignment value between each base in each codon sequence with the corresponding base in the target motif in the 5′ to 3′ direction is determined.

The matching value of each codon sequence with the target motif is obtained based on the alignment value of each base in each codon sequence.

Illustratively, when the target motif may correspond to at least one selected from (a1) to (a6): (a1) TTGGGTCTX; (a2) XTTGGGTCT; (a3) XTCTGGGTT; (a4) TCTGGGTTX; (a5) XXTTGGGTCTXX; (a6) XXTCTGGGTTXX, where X=A, T, C, G. The traversing m codon sequence sets to obtain a matching value of each codon sequence in each of the codon sequence sets with a target motif includes:

S31: by taking (a1) TTGGGTCTX as the target motif, tranversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with target motif. For a codon sequence, the 6th position therein must be base “T”, and the 7th position therein must be base “C”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S32: by taking (a2) XTTGGGTCT as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 7th position therein must be base “T”, and the 8th position therein must be base “C”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S33: by taking (a3) XTCTGGGTT as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 3th position therein must be base “C”, and the 4th position therein must be base “T”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S33: by taking (a3) XTCTGGGTT as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 3th position therein must be base “C”, and the 4th position therein must be base “T”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S34: by taking (a4) TCTGGGTTX as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 3rd position therein must be base “T”, and the 2nd position therein must be base “C”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S35: by taking (a5) XXTTGGGTCTXX as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 7th position therein must be base “T”, and the 8th position therein must be base “C”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

S36: by taking (a6) XXTCTGGGTTXX as the target motif, traversing each codon sequence of the codon sequence sets C₁-C_(m) to calculate the matching value of each codon sequence with the target motif. For a codon sequence, the 4th position therein must be base “C”, and the 5th position therein must be base “T”, or the matching value is not outputted. When the codon sequence contains an effective base pair, the bases at positions 1 to 9 are successively aligned, and scores are accumulated according to the alignment results.

In some embodiments, the obtaining a matching value of each codon sequence in each of the codon sequence sets with a target motif further includes: determining whether each codon sequence in each codon sequence set is hybridized with an intron sequence to obtain a complementary value of each codon sequence in each of codon sequence sets.

Illustratively, by traversing each codon sequence of the codon sequence sets C₁-C_(m), based on whether each codon sequence is hybridized with an intron sequence, the complementary value of each codon sequence is obtained; and in combination of the complementary value with the alignment value, a final matching value of each codon sequence is obtained.

S4, determining the target codon sequence in the codon sequence set based on the matching value, where a position of the target codon sequence corresponding to the coding region sequence is an implantation position of the ribozyme recognition site, and the coding region sequence containing the target codon sequence at the implantation position is the target coding region sequence containing the ribozyme recognition site.

Specifically, after obtaining the matching value of each codon sequence in each of codon sequence sets with a target motif, the codon sequence with the matching value higher than a first threshold is selected as the target codon sequence. The position of the target codon sequence corresponding to the coding region sequence is the implantation position of ribozyme recognition site. For the target coding region sequence, the nucleotide sequence at the implantation position is the target codon sequence.

Furthermore, the target codon sequence is truncated at the position of the effective base pair in the target codon sequence, to provide the truncated fragment I of the coding element and the truncated fragment II of the coding element, or provide the truncated fragment III of the coding element and the truncated fragment IV of the coding element, either of which may be applied to the Clean PIE system for circularization.

In the present disclosure, the “first threshold” refers to a value that when the matching value between the codon sequence and the target motif is higher than this value, the truncated fragments of the coding element may be applied to the Clean PIE system for effective circularization.

In the present disclosure, as a limited number of base mutations in the ribozyme recognition site sequence have no impact on effective circularization of the ribozyme recognition site, for the target codon sequence, mutant bases not matching the target motif are allowed at certain positions.

Methods for Screening Ribozyme Recognition Sites

In some embodiments, the present disclosure provides a method for screening a target coding region sequence containing a ribozyme recognition site, the method including the following steps:

(1) Determining a sequence to be screened, which contains an intron sequence derived from Group I intron, a first exon sequence ligated to the 5′ end of the intron sequence, and a second exon sequence ligated to the 3′ end of the intron sequence.

Specifically, the sequence to be screened may be any type of sequence with self-splicing activity of Group I intron.

(2) Obtaining a predicted RNA secondary structure based on the sequence to be screened.

Specifically, the obtaining the RNA secondary structure of the sequence to be screened includes:

-   -   receiving, by a sequence alignment software (for example,         ClustalW), an input of a sequence to be screened, to determine         highly conserved sequences P7 and P7′ in the sequence to be         screened derived from the Group I intron, and determine J6/7 and         J8/7 sequences to obtain first pairing information;     -   determining P3, and determining whether a P3′ sequence exists         after P7, and if not, adding a P3′ sequence, to obtain second         pairing information;     -   determining one or more of the following sequences: P2, P4, P5,         P6, P8, and P9, according to the first pairing information and         the second pairing information, and further according to a         typical structure of the Group I intron (FIG. 34 );     -   determining P1′ and P10 together with the ribozyme recognition         site by a first exon sequence at the 5′ end and a second exon         sequence at the 3′ end; and predicting by the method based on         minimum free energy in Mfold or RNAstructure using the obtained         pairing information to obtain the predicted RNA secondary         structure.

(3) Obtaining a ribozyme recognition site I with ribozyme recognition activity in the first exon sequence and a ribozyme recognition site II with ribozyme recognition activity in the second exon sequence based on the predicted RNA secondary structure.

In some embodiments, the nucleotide sequence of the ribozyme recognition site I is hybridized with a leader sequence in the intron sequence, or the nucleotide sequence of the ribozyme recognition site II is hybridized with a leader sequence in the intron sequence. The nucleotide sequence of the ribozyme recognition site I and/or the ribozyme recognition site II are hybridized with the leader sequence in the intron sequence, keeping two ends of the Clean PIE system close to each other during self-splicing to form a circular RNA, which facilitates successive cleavage and ligating at positions of the ribozyme recognition site I and the ribozyme recognition site II to form the circular RNA.

As a limited number of base mutations in the ribozyme recognition site sequence have no impact on effective circularization of ribozyme recognition site, in some embodiments, the bases on the ribozyme recognition site I are successively replaced to obtain a mutant sequence of the ribozyme recognition site I with ribozyme recognition activity. In some embodiments, the bases on the ribozyme recognition site II are successively replaced to obtain a mutant sequence of the ribozyme recognition site II with ribozyme recognition activity.

(4) Determining the ribozyme recognition site sequence based on the nucleotide sequence of the ribozyme recognition site I and the nucleotide sequence of the ribozyme recognition site II. Specifically, the ribozyme recognition site sequence contains at least one selected from the group consisting of (i) to (iv):

-   -   (i) the nucleotide sequence of the ribozyme recognition site I,     -   (ii) the nucleotide sequence of the ribozyme recognition site         II,     -   (iii) a mutant sequence of the ribozyme recognition site I with         ribozyme recognition activity,     -   (iv) a mutant sequence of the ribozyme recognition site II with         ribozyme recognition activity.

In some embodiments, the ribozyme recognition site contains (i) the nucleotide sequence of the ribozyme recognition site I, and (ii) the nucleotide sequence of the ribozyme recognition site II. In other embodiments, the ribozyme recognition site consists of (i) the nucleotide sequence of the ribozyme recognition site I and (ii) the nucleotide sequence of the ribozyme recognition site II.

In some embodiments, the ribozyme recognition site contains (iii) a mutant sequence of the ribozyme recognition site I with ribozyme recognition activity, and (iv) a mutant sequence of the ribozyme recognition site II with ribozyme recognition activity. In other embodiments, the ribozyme recognition site consists of (iii) a mutant sequence of the ribozyme recognition site I with ribozyme recognition activity and (iv) a mutant sequence of the ribozyme recognition site II with ribozyme recognition activity.

In some embodiments, the ribozyme recognition site contains (i) the nucleotide sequence of the ribozyme recognition site I, and (iv) a mutant sequence of the ribozyme recognition site II with ribozyme recognition activity. In other embodiments, the ribozyme recognition site consists of (i) the nucleotide sequence of the ribozyme recognition site I and (iv) a mutant sequence of the ribozyme recognition site II with ribozyme recognition activity.

In some embodiments, the ribozyme recognition site contains (iii) a mutant sequence of the ribozyme recognition site I with ribozyme recognition activity, and (ii) the nucleotide sequence of the ribozyme recognition site II. In other embodiments, the ribozyme recognition site consists of (iii) a mutant sequence of the ribozyme recognition site I with ribozyme recognition activity, and (ii) the nucleotide sequence of the ribozyme recognition site II.

Screening System for Screening Target Coding Region Sequence Containing Ribozyme Recognition Site

In some embodiments, the present disclosure provides a screening system for screening a target coding region sequence containing a ribozyme recognition site, which includes:

a target motif constructing module configured to ligate x nucleotide to at least one end of the 5′ end and the 3′ end of a ribozyme recognition site sequence to obtain a target motif with 3n nucleotide; where each x is an integer≥0 independently of one another, and each ligated nucleotide is selected from any type of nucleotide independently of one another;

an amino acid unit extraction module configured to extract m amino acid units from the target polypeptide containing q amino acids in the N-terminal-to-C-terminal direction, with each of the amino acid units containing n amino acids; where at least one repeated amino acid is contained between any two adjacent amino acid units, n is an integer and n≥2, m is an integer and m≥1; preferably, m=q+1-n;

-   -   a codon sequence set extraction module configured to determine m         codon sequence sets, where each of the codon sequence sets         includes a codon sequence corresponding to each of the amino         acid units;     -   a matching value calculation module configured to traverse the m         codon sequence sets to obtain a matching value of each codon         sequence in each of the codon sequence sets with the target         motif;     -   a target codon sequence screen module configured to determine         the target codon sequence in the codon sequence set based on the         matching value, where a position of the target codon sequence         corresponding to the coding region sequence is an implantation         position of the ribozyme recognition site, and the coding region         sequence containing the target codon sequence at the         implantation position is the target coding region sequence         containing the ribozyme recognition site.

In some embodiments, the matching value calculation module includes:

-   -   an effective base pair determination unit configured to         determine whether a base at the position corresponding to an         effective base pair in the codon sequence is an effective base,         and if the codon sequence does not contain the effective base         pair, disable output of the alignment value of the codon         sequence;     -   an alignment value determination unit configured to determine         the alignment value between each base in each codon sequence         with the corresponding base in the target motif in the 5′ to 3′         direction;     -   a matching value output unit configured to obtain the matching         value of each codon sequence with the target motif based on the         alignment value of each base in each codon sequence.

In some embodiments, the matching value calculation module further includes:

-   -   a complementary value calculation module configured to determine         whether each codon sequence in each of the codon sequence sets         is hybridized with an intron sequence to obtain the         complementary value of each codon sequence in each of the codon         sequence sets;     -   the matching value output unit configured to determine the         matching value of each codon sequence in each of the codon         sequence sets with the target motif based on the alignment value         and the complementary value.

Moreover, the present disclosure also discloses a processing device for screening a target coding region sequence containing a ribozyme recognition site, which includes:

-   -   a memory configured to store computer programs;     -   a processor configured to execute a computer program to         implement the above method for screening a target coding region         sequence containing a ribozyme recognition site.

Moreover, the present disclosure also discloses a computer-readable storage medium, on which a computer program is stored, which, when executed by a processor, implements the above method for screening a target coding region sequence containing a ribozyme recognition site.

It may be further appreciated by those skilled in the art that the units and algorithm steps of each example described in connection with the embodiments disclosed herein may be implemented by electronic hardware, computer software, or a combination thereof. In order to clearly illustrate the interchangeability of hardware and software, components and steps of each example have been generally described in terms of functions in the above description.

Whether these functions are performed by hardware or software depends on the specific application and design constraints of the technical scheme. Those skilled in the art may use different methods to achieve the described functions for each specific application, which should not be construed as beyond the scope of the present disclosure.

EXAMPLES

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating the embodiments of the present disclosure) are given only for purposes of illustration, as various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading the detailed description.

Unless otherwise specified, the experimental techniques and methods used in the examples herein are conventional ones, such as those without specific conditions in the following examples, which are carried out in accordance with conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or those suggested by manufacturers. The materials and reagents used are commercially available, unless otherwise specified.

Example 1: Matching Value Calculation Module

This example illustrates the scoring cateria of matching value calculation module in the screening system for target coding region sequences. The scoring principle of the matching calculation module is intended to find a target codon sequence closest to Group I intron PIE E1E2 in the ORF (open reading frame) of the circularization gene according to the degeneracy principle of encoding amino acids, thereby determining a ribozyme recognition site in the coding region.

If there was no target codon sequence completely matching to the Group I intron E1E2 in the coding region, the scoring was performed according to the following principles:

1. The completely matched sequence was taken as the baseline and given a score of 100. By taking T4td PIE as an example, the scoring cateria are shown in FIG. 16 :

The score was 17.6 if the 1st position of the sequence was “T”, or it was 0. If the 2nd position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 3rd position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8 respectively, or by 0. If the 4th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. if the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 8th position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0. Finally, the total score was the scoring of the sequence.

2. The E1E2 sequences were counted, and the bases (effective base pair) at the circularization site were excluded, which were not scored (taking T4td PIE as an example, where E1E2 sequence was TTGGGTCT and TC was the circularization site, then TC was not scored).

Weighted average was carried out according to the following four situations:

-   -   a) if the sequence in the coding region completely matched the         E1E2 sequence, and the circularization position sequence in the         coding region was base complementary to IG sequence (a leader         sequence) in the intron, then the weighted average was 3;     -   b) if the sequence in the coding region completely matched the         E1E2 sequence, whereas the circularization position sequence in         the coding region was not base complementary to IG sequence (a         leader sequence) in the intron, then the weighted average was 2;     -   c) if sequences in the coding region cannot match E1E2 sequence,         whereas the circularization position sequence in the coding         region was base complementary to IG sequence (guide sequence) in         the intron, then the weighted average was 1; and     -   b) if sequences in the coding region cannot match the E1E2         sequence, and the circularization position sequence in the         coding region was not base complementary to IG sequence (guide         sequence) in the intron, then the weighted average was 0.

The circularization sequence may be scored based on the above principles.

The present circularization sequence scoring system may be applied to Example 2 to obtain the target codon sequence, which is the same as or similar to the Group I intron PIE E1E2 sequence.

Example 2: Screening for Target Coding Region Sequence Containing Ribozyme Recognition Site

FIG. 17 shows an automated flow chart for determining a target containing a ribozyme recognition site using the screening system, which was performed as follows:

(1) converting the coding region of a complete gene sequence into an amino acid sequence;

(2) in the amino acid unit extraction module, performing a slide window on the amino acid sequence, with a step of 1 (step=1) and a window length of 3 or 4 (win=3, 4), to obtain a short amino acid sequence with a length of 3 or 4;

(3) in the codon sequence set extraction module, translating the short amino acid sequence obtained in (2) into a nucleotide sequence according to the codon table in (1) to obtain a codon sequence set; and

(4) in the matching value calculation module, scoring the codon sequence, with the E1E2 sequence as the ribozyme recognition site being TGGGTCT and taking the following sequences as the target motif, respectively, successively calculating the matching values of the codon sequences in the codon sequence sets with the target motif, where X=A, T, C, G.

TTGGGTCTX; XTTGGGTCT; XTCTGGGTT; TCTGGGTTX; XXTTGGGTCTXX; XXTCTGGGTTXX.

i) The sequences obtained in (3) were traversed (win=3, with a sequence length of 9 bp) and scored based on TTGGGTCTX (X=A, T, C, G), and the 6th position of the sequence must be the base “T” and the 7th position must be the base “C”. If the 1st position of the sequence was “T”, the score was 17.6, or it was 0. If the 2nd position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 3rd position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 4th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 8th position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0.

ii) The sequences obtained in (3) were traversed (win=3, with a sequence length of 9 bp) and scored based on XTTGGGTCT (X=A, T, C, G), and the 7th position of the sequence must be the base “T” and the 8th position must be the base “C”. If the 2nd position of the sequence was “T”, the score was 17.6, or it was 0. If the 3rd position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 4th position of the sequence was “G” and “A” the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 6th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 9th position of the sequence was “T” and “A″”, the score was accumulated by 12 and 5.8, respectively, or by 0.

iii) The sequences obtained in (3) were traversed (win=3, with a sequence length of 9 bp) and scored based on XTCTGGGTT (X=A, T, C, G), and the 3rd position of the sequence must be the base “C” and the 4th position must be the base “T”, If the 9th position of the sequence was “T”, the score was 17.6, or it was 0. If the 8th position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 7th position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 6th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 2nd position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0.

iv) The sequences obtained in (3) were traversed (win=3, with a sequence length of 9 bp) and scored based on TCTGGGTTX (X=A, T, C, G), and the 3rd position of the sequence must be the base “T” and the 2nd position must be the base “C”. If the 8th position of the sequence was “T”, the score was 17.6, or it was 0. If the 7th position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 6th position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 4th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 1st position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0.

v) The sequences obtained in (3) were traversed (win=4 with a sequence length of 12 bp) and scored based on XXTTGGGTCTXX (X=A, T, C, G), and the 7th position of the sequence must be the base “T” and the 8th position must be the base “C”. If the 1st position of the sequence was “T”, the score was 17.6, or it was 0. If the 3rd position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 4th position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 5th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 6th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 9th position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0.

vi) The sequences obtained in (3) were traversed (win=4, with a sequence length of 12 bp) and scored based on XXTCTGGGTTXX (X=A, T, C, G), and the 4th position of the sequence must be the base “C” and the 5th position must be the base “T”. If the 10th position of the sequence was “T”, the score was 17.6, or it was 0. If the 9th position of the sequence was “T” and “C”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 8th position of the sequence was “G” and “A”, the score was accumulated by 17.6 and 5.8, respectively, or by 0. If the 7th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 6th position of the sequence was “G”, the score was accumulated by 17.6, or by 0. If the 3rd position of the sequence was “T” and “A”, the score was accumulated by 12 and 5.8, respectively, or by 0.

(4) Sorting according to the sequence score from high to low.

By taking EGFP protein as an example, the nuclease recognition site in the nucleotide sequence encoding EGFP protein was determined through the following steps.

i) EGFP nucleotide sequence seq1 (SEQ ID NO: 1) was translated into amino acid sequence seq 2 (SEQ ID NO: 2).

ii) A slide window was performed: (a) splitting the seq2 sequence with a step of 1 (step=1) and a window length of 3 to obtain amino acid fragments with a length of three amino acids, randomly recombining different codons corresponding to each amino acid fragment to obtain possible base sequences corresponding to all amino acid fragments by exhaustion, and constructing a codon sequence set; and (b) splitting the seq2 sequence with a step of 1 (step=1) and a window length of 4 to obtain amino acid fragments with a length of 4 amino acids, randomly recombining different codons corresponding to each amino acid fragment, to obtain possible base sequences corresponding to all amino acid fragments by exhaustion, and adding the corresponding base sequences into the constructed codon sequence set above.

Illustratively, the seq2 sequence is split with a step of 1 and a window length of 3 amino acids, to obtain the amino acid fragments shown below:

MVS, VSK, SKG, KGE, GEE, ...... LYK, YK*;

-   -   where the above “ ” represents the amino acid fragments between         GEE and LYK obtained by successively splitting the seq2 sequence         with a step of 1 and a window length of 3 amino acids, and the         “*” stands for a stop codon.

The seq2 sequence was split with a step of 1 and a window length of 4 amino acids, to obtain the amino acid fragments shown below:

(SEQ ID NO: 60) MVSK, (SEQ ID NO: 61) VSKG, (SEQ ID NO: 62) SKGE, ...... (SEQ ID NO: 63) ELYK, LYK*;

-   -   where the above “ ” represents the amino acid fragments between         SKGE and ELYK obtained by successively splitting the seq2         sequence with a step of 1 and a window length of 4 amino acids,         and the “*” stands for a stop codon.

iii) The codon sequence set constructed in (2) were tranversed, scoring was performed by taking TTGGGTCTX, XTTGGGTCT, XXTTGGGTCTXX, TCTGGGTTX, XTTGGTTT and XXTCTGGGTTXX as target motifs, which were used as the matching values of codon sequences with the target motif; and the sequence scores were sorted from high to low, as shown in Table 1, where the “ ” represents the traversed codon sequences in the codon sequence set and corresponding matching values.

TABLE 1 seq score SEQ ID NO: XXTTGGGTCTXX SEQ ID NO: 64 TTTTCGGTCTCT 82.4 SEQ ID NO: 65 ATTTTGGTCGAG 76.2 SEQ ID NO: 66 ACTTTGGTCAAT 76.2 SEQ ID NO: 67 GGTTCGGTCCAG 76.2 SEQ ID NO: 68 TTTTCGGTCAGT 76.2 SEQ ID NO: 69 ACTGGGGTCGTT 76.2 SEQ ID NO: 70 TATGGGGTCCAG 76.2 SEQ ID NO: 71 ...... ...... XTTGGGTCT GATGGGTCT 82 GATGGGTCG 76.2 GATGGGTCC 76.2 GATGGGTCA 76.2 ...... ...... TTGGGTCTX TTGGGTCAT 93.8 TTAGGTCAT 82 CTGGGTCAT 76.2 ...... ...... XXTCTGGGTTXX SEQ ID NO: 72 ATTCTGGGTCAT 82 SEQ ID NO: 73 ACTCTGGGTATG 82 SEQ ID NO: 74 ACGCTGGGTATG 76.2 SEQ ID NO: 75 ATCCTGGGTCAT 76.2 SEQ ID NO: 76 ACCCTGGGTATG 76.2 SEQ ID NO: 77 ATACTGGGTCAT 76.2 SEQ ID NO: 78 ACACTGGGTATG 76.2 SEQ ID NO: 79 ...... ...... XTCTGGGTT ATCTTGGTT 82 TTCTCGGTT 82 GGCTCGGTT 76.2 ACCTTGGTT 76.2 ...... ...... TCTGGGTTX TCTGCGTTG 82 GCTGGGATT 76.2 CCTGTGTTG 76.2 ACTGGGGTT 76.2 ...... ......

iv) The target codon sequence was obtained according to the calculation results of matching values in step (3), wherein the coding region sequence containing the target codon sequence was the target coding region sequence.

Example 3: In Vitro Synthesis of Circular mRNA Encoding eGFP

This example provides a method for preparing a circular mRNA capable of expressing eGFP by the coding region sequence of eGFP containing ribozyme recognition site screened by the present disclosure.

(1) Screening of eGFP Gene Truncation Site and Plasmid Construction

DGS, the amino acid unit to be optimized obtained by the method provided in Example 1-2, was codon-optimized to GAT GGA TCA (ribozyme recognition site sequence) and truncated by TC (effective base pair) to form a structure in the form of T4td intron fragment II-eGFP truncated fragment II-ev29-eGFP truncated fragment I-T4td intron fragment I, and the amino acids and nucleotide sequences involved in this example were shown in Table 2 below.

TABLE 2 SEQ ID NO: eGFP protein sequence: SEQ ID NO: 3 Intron fragment II SEQ ID NO: 4 Intron fragment I SEQ ID NO: 5 Ev29 sequence SEQ ID NO: 10 Untruncated eGFP sequence SEQ ID NO: 11 eGFP truncated fragment I sequence SEQ ID NO: 12 eGFP truncated fragment II sequence SEQ ID NO: 13

Plasmid synthesis and cloning under this architecture was commissioned to Suzhou GENEWIZ, from Azenta Life Sciences Suzhou, China. The resultant gene fragment was ligated to pUC57 vector. The resultant plasmid was as follows: pUC57-EV29-eGFP (SEQ ID NO: 14).

(2) Preparation of Linear Plasmid Template

1) Plasmid Extraction

i) The stab cultured bacteria custom-synthesized were activated under conditions of 37° C./220 rpm/3-4 h.

ii) The activated bacterial solution was taken for amplification culture under culture conditions of 37° C./220 rpm/overnight.

iii) The plasmids were extracted (with TIANGEN® EndoFree Mini Plasmid Kit II), and then the OD value thereof was measured.

2) Plasmid Enzyme Digestion

The plasmid prepared in the above step 1) was digested by XbaI single enzyme digestion method, and the digestion system was shown in the following table:

TABLE 3 Reagent Volume Plasmid 10 μg Enzyme (1000 units) 5 μL 10 × cutsmart buffer 50 μL Nuclease free, H₂O Total 500 μL

Enzyme digestion was carried out at 37° C. overnight. The enzyme digestion products were recovered by universal DNA gel recovery kit (Universal DNA Purification Kit, TIANGEN BIOTECH CO., LTD.), and then the OD value thereof was measured. After that, the restriction enzyme products were identified by 1% agarose gel electrophoresis. The purified linear plasmid template was used for in vitro transcription.

(3) In Vitro Transcription for Preparing Linear mRNA

1) In Vitro Transcription

mRNA was synthesized by T7 in vitro transcription kit (APExBIO T7 High Yield RNA Synthesis Kit), and the transcription system was shown in the following table.

TABLE 4 Reagent Volume 10 × Reaction Buffer 2 μL ATP (20 mM) 2 μL CTP (20 mM) 2 μL IKA (20 mM) 2 μL GTP (20 mM) 2 μL Linearized DNA template 1 μg T7 RNA Polymerase Mix 2 μL RNA Nuclease free, H₂O Total 20 μL

The resultant was incubated at 37° C. for 2.5 h. Then, the entire product was incubated for 15 min at 37° C. to digesting the linearized DNA template by DNase I.

2) Purification of Linear mRNA

The transcript obtained from step 1) above was purified by silica-based membrane technology in the form of a spin column (Thermo, Gene Jet RNA Purification Kit), and then the OD value thereof was measured. Further, the RNA size was identified by 1% denaturing agarose gel electrophoresis.

3) Purification of Linear mRNA

The transcript obtained from the above step 1) was purified by silica-based membrane technology in the form of a spin column (Thermo, Gene Jet RNA Purification Kit), and then the OD value thereof was measured. Further, the RNA size was identified by 1% denaturing agarose gel electrophoresis.

The method for preparing 1% denaturing agarose gel includes:

-   -   1) 1 g of agarose was weighed into 72 mL of nuclease-free H₂O         and heated until dissolved in a microwave oven.     -   2) After the above agarose was cooled to 55-60° C., 0.1% gel         red, 10 mL of 10×MOPS, and 18 mL of formaldehyde were added         therein in the fume hood. Then, pouring gel was carried out.     -   3) The procedure of denaturing agarose gel electrophoresis         includes: the sample RNA and 2× Loading buffer were taken in         equal volume, and denaturated at 65-70° C. for 5 to 10 min. The         sample was loaded and electrophoresed at 100 V/30 min, and then         photographed by gel imaging system.

(4) Circularization of mRNA

1) Circularization Reagent:

GTP Buffer: 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, about pH 7.5.

2) Circularization System and Conditions:

TABLE 5 Solution Volume mRNA 25 μg mRNA GTP solution (20 mM) 50 μL GTP buffer up to 500 μL

The above solution was heated at 55° C. for 15 min and then placed on ice, the circularized RNA products were purified by silica-based membrane technology in the form of a spin column (Thermo, Gene Jet RNA Purification Kit), and then the OD value thereof was measured. Further, the RNA size was identified by 1% denaturing agarose gel electrophoresis.

3) Identification of Circular RNA by 1% Denaturing Agarose Gel

Reagent preparation: 1 g agarose powder was added into 72 mL of nuclease-free water and heated until dissolved, then 10 mL of 10×MOPS buffer solution was added. Then 18 mL of fresh 37% formaldehyde was added in the fume hood and mixed thoroughly, and the gel was poured into the tank.

mRNA detection: About 500 ng of mRNA solution was taken for mixing with 2×RNA loading buffer in equal volume, followed by heating at 65° C. for 5 min. Further, agarose gel detection was carried out.

Experimental Results:

TABLE 6 ENZYME TRANSCRIP- DNASEI PLASMID-1 CUT TION DIGEST CIRCULIZATION Con. Con. Con. Con. Con. (ng/μL) Vol. (ng/μL) Vol. (ng/μL) Vol. (ng/μL) Vol. (ng/μL) Vol. Classic PIE 313 105 μL 162.2 50 μL 2372 70 μL 2007.5 55 μL 248 35 μL EGFP (Clean 329 105 μL 128.8 50 μL 3992 70 μL 1725.3 55 μL 269.2 35 μL PIE) of the present disclosure

classic FIG. 18 shows detection results of agarose gel electrophoresis of the digested products (A) of the plasmids used for the preparation of circular mRNA by using a classic PIE system and a Clean PIE system of the present disclosure respectively, and that of the circularized products (B).

The above results showed that compared with the classic PIE system, the circularization process and technology of preparing circular RNAs using the Clean PIE system in the present disclosure involved no additional changes, showing obvious circularization effect. The circularization efficiency using the Clean PIE system was found similar to that using the classic PIE system through agarose gel electrophoresis, with no obvious difference found.

Example 4: Verification of In Vitro Expression of Circular mRNAs Synthesized In Vitro by the Method of the present disclosure

In this example, the circular mRNAs prepared in Example 3 were delivered into 293T cells, and the expression in 293T cells of the circular mRNAs synthesized in vitro by the present method was detected, with the following specific process:

(1) Cell Culture

293T cells were inoculated into DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in an incubator containing 5% CO₂ at 37° C. Cells were subcultured every 2 to 3 days.

(2) Cell Transfection

Before transfection, 293T cells were inoculated into a 24-well plate with 1×10⁵ cells/well, and cultured in an incubator containing 5% CO₂ at 37° C. After the cells reached 70-90% confluence, mRNAs were transfected into 293T cells by Lipofectamine MessengerMax (Invitrogen) transfection reagent at 500 ng/well, according to the following specific process:

1) Messenger MAX™ Reagent was diluted with a dilution ratio shown in the following table.

TABLE 7 Reagent Volume/well MEM serum-free medium 25 μL MessengerMAX ™ Reagent 0.75 μL

After diluting and mixing, the solution was allowed to stand at room temperature for incubating for 10 min.

2) mRNAs were diluted with a dilution ratio shown in the following table:

TABLE 8 Reagent Volume/well mRNA 1 μg MEM serum-free medium up to 25 μL

3) The mixed and diluted Messenger MAX™ Reagent and mRNA (1:1) as shown in the following table was taken.

TABLE 9 Reagent Volume/well Diluted MessengerMAX ™ Reagent 25 μL Diluted mRNA 25 μL

After diluting and mixing, the solution was allowed to stand at room temperature for incubating for 5 min.

4) 50 μL of the above mixture was taken up, and slowly added into a 24-well plate adhering to the wall, then incubated in an incubator containing 5% CO₂ at 37° C.

(3) Protein expression detection:

1) Fluorescence observation of cells: 24 hours after transfection, the expression of EGFP was observed in 293T cells under a 200×fluorescence microscope.

2) Detecting the average fluorescence intensity of cells by flow cytometry: the average fluorescence intensity of 293T cells 24 hours after transfection was detected by flow cytometry.

FIGS. 19A and 19B shows the detection results of in vitro expression level of circular mRNAs prepared by the classic PIE system and the Clean system of the present disclosure, where A: the observation results of fluorescence microscope, and B: the detection results of flow cytometry.

The results in FIGS. 19A and 19B showed as follows. The present disclosure unexpectedly found that compared with the circular mRNAs prepared by classic PIE circularization method, the cellular fluorescence of the circular mRNAs prepared by Clean PIE of the present disclosure was strongly enhanced in 293T transfected cells, indicating that the circular mRNAs prepared by Clean PIE of the present disclosure allowed for improved stability of the circular mRNAs in cells. Thus no substantial immunogenicity is occurred, because no additional exon sequences were introduced. Meanwhile, the flow cytometry data also showed that in vitro expression level of the circular mRNAs prepared by Clean PIE of the present disclosure was unexpectedly higher than that of the circular mRNAs prepared by classic PIE. In general, the above results indicated that the Clean PIE system of the present disclosure can improve the expression level of circular mRNAs on the basis of obtaining more accurate circular mRNAs.

Example 5: Application of Clean PIE System for Different Proteins

In this example, many circularization applications for a variety of other different proteins were prepared by the Clean PIE system. The coding region sequences involved in the present disclosure included spCas9, firefly Luciferase, IL12, and FLAG-con1-SPOP167-274, as follows:

1) DGS, the amino acid unit to be optimized of spCas9 obtained by the method provided in Examples 1-2, was codon-optimized to GAT GGA TCA (ribozyme recognition site sequence) and truncated by TC (effective base pair) site to form a structure in the form of T4td intron fragment II-spCas9 truncated fragment II-ev29-spCas9 truncated fragment I-T4td intron fragment I;

2) LRS, the amino acid unit to be optimized of firefly Luciferase obtained by the method provided in Examples 1-2, was codon-optimized to CTT AGG TCT (ribozyme recognition site sequence) and truncated by TC (effective base pair) site to form a structure in the form of T4td intron fragment II-fLUC truncated fragment II-ev29-fLUC truncated fragment I-T4td intron fragment I;

3) LGS, the amino acid unit to be optimized of IL12 obtained by the method provided in Examples 1-2, was codon-optimized to CTT GGG TCT (ribozyme recognition site sequence) and truncated by TC (effective base pair) site to form a structure in the form of T4td intron fragment II-IL12 truncated fragment II-ev29-IL12 truncated fragment I-T4td intron fragment I;

4) LGP, the amino acid unit to be optimized of FLAG-con1-SPOP167-274 obtained by the method provided in Examples 1-2, was codon-optimized to TTG GGT CCT (ribozyme recognition site sequence) and truncated by TC (effective base pair) site to form a structure in the form of T4td intron fragment II-FLAG-con1-SPOP167-274 truncated fragment II-ev29-FLAG-con1-SPOP167-274S truncated fragment I-T4td intron fragment I.

Circular mRNAs expressing spCas9, firefly Luciferase, IL12 and FLAG-con1-SPOP167-274 were prepared, respectively, by the experimental method in Example 3, and the sequences involved in this example were shown in the following table.

TABLE 10 SEQ ID NO: spCas9 truncated fragment I SEQ ID NO: 15 spCas9 truncated fragment II SEQ ID NO: 16 fLUC truncated fragment I SEQ ID NO: 17 fLUC truncated fragment II SEQ ID NO: 18 IL12 truncated fragment I SEQ ID NO: 19 IL12 truncated fragment II SEQ ID NO: 20 FLAG-con1-SPOP167-274 truncated SEQ ID NO: 21 fragment I FLAG-con1-SPOP167-274 truncated SEQ ID NO: 22 fragment II

FIG. 20 showed the detection results of agarose gel electrophoresis of circular mRNAs expressing different proteins prepared by circularization of Clean PIE. The experimental results showed that as found from circularization of different proteins (spCas9, firefly Luciferase, IL12, and FLAG-con1-SPOP167-274), after the ribozyme recognition site sequence was obtained by optimization in different proteins, the proteins using the Clean PIE circularization system of the present disclosure each showed difference in their migration rates in agarose gel after circularization. Thus, the results demonstrated that the coding sequences of all proteins can be effectively circularized. In general, the above results indicated that the method provided in the present disclosure can be effectively applied between proteins with different sequences, exhibiting good universality and compatibility, and can be used as a novel macromolecule (≥1000 bp) circularization method.

Example 6: Analysis of Applicability of Clean PIE System

In this example, the availability of the Clean PIE system of the present disclosure in the genes of Escherichia coli genome was verified and analyzed by way of bioinformatics. The results indicated that the method of searching for the target circularization sequence (the sequence containing ribozyme recognition site) by the present disclosure has universal applicability.

By taking T4td PIE system as an example: bioinformatics evaluation of genes over 1000 bp and 500 bp in the genome sequence of Escherichia coli was performed to screen the effective circularization sequences (score≥70).

FIG. 21 shows the evaluation results of scoring the matching values for genes over 1000 bp and 500 bp in the genome of Escherichia coli. The results showed as follows.

By placing the genes of Escherichia coli genome over 1000 bp and 500 bp into the screening system, and performing evaluation using the matching value calculation module, it was found that the probability of finding the target circularization sequence with a score of 82 or more in the genes over 500 bp reached 100%. In addition, this probability can be further improved by combining E1E2 sequences of different Group I introns for the coding gene. That is, the most suitable intron and its corresponding E1E2 sequence were screened by the matching value calculation module as the circularization site (ribozyme recognition site) of circular mRNA.

Example 7: RNaseR Validation of Circularization

In this example, the feasibility of the circularization method of the present disclosure was verified by digesting linear and circular mRNAs. Specifically, as compared with linear RNAs, circular RNAs have better tolerance to RNaseR, thus the sequence circularization can be verified by comparing the tolerance to RNaseR of linear mRNAs and circular mRNAs circularized by Clean PIE.

On the basis of Example 3, the linear and circular mRNAs derived from classic PIE system and Clean PIE system, respectively, were digested by RNase, which was purchased from MClab with ITEM NO. RNASR-100, with the RNaseR digestion system as follows:

TABLE 11 mRNA 1 μg RNase R 1 U 10 × Reaction buffer 2 μL H₂O Up to 20 μL

Incubation was carried out at 37° C. for 5 min, and then inactivation was carried out by incubating at 70° C. for 5 min.

FIG. 22 shows RNaseR digestion of the linear and circular mRNAs produced by different PIE systems. The results showed that circular mRNAs, whether circularized by classic PIE or Clean PIE system, showed good tolerance to RNaseR, indicating that the Clean PIE system can effectively form circular mRNAs.

Example 8: Verification of Clean PIE System Circularization Method by Capillary Electrophoresis

In this example, the prepared circular mRNAs were detected by capillary electrophoresis.

The concentration of crude products of circular RNA obtained by circularization in Example 7 were measured by micro-spectrophotometry (Nano-Drop Technologies, Thermo). Then, the crude products were treated by RNA 6000 Assay Kit (Agilent 5067-1511), detected and analyzed by Agilent 2100 automated nucleic acid analyzer. The procedures for the kit and analyzer can be found in Agilent's official website.

FIGS. 23A through 23C shows the analysis results of capillary electrophoresis. The top figure (FIG. 23A) was the capillary electrophoresis detection pattern of the circular RNA products without RNaseR treatment and HPLC purification, the middle figure (FIG. 23B) was the partial enlargement of the top figure, and the bottom figure (FIG. 23C) was the molecular weight standard. The results showed as follows.

By capillary electrophoresis, the circularized RNA showed single peak, and had two intron peaks at nt-218 and nt-243, and one intron dimer peak at nt-349. The total circular mRNAs accounted for 89.6% (before purification), further indicating that the method of the present disclosure can effectively realize the circularization of circular messenger ribonucleotides.

Example 9: Verification of Sequence Integrity and Sequence Accuracy at Circularization Site (Ribozyme Recognition Site)

In this example, the sequence integrity and accuracy at the circularization site of the circular mRNA circularized by the circularization method of the invention were identified by sequencing after reverse transcription. The primer sequences used in this example were as follows:

Fluc-R: (SEQ ID NO: 23) TACTTGTCGATCAGGGTGCT Fluc-F: (SEQ ID NO: 24) TGGACAGCAAGACCGACTAC IL12-R: (SEQ ID NO: 25) CTGCATCAGCTCGTCGATGG IL12-F: (SEQ ID NO: 26) TACTACAACAGCAGCTGCAGCA

Each of the linear and circular mRNAs of firefly Luciferase (Flue) and IL12 in Example 5 was reverse transcribed into a first strand cDNA by the reverse transcription kit of Takara Company (RR037B, Takara). By using cDNA as a template, PCR amplification was carried out with specific primers to obtain amplified fragments. The sequence of the actually synthesized circular mRNA was compared with that of the designed circular mRNA by sequencing.

FIGS. 24A and 24B shows the analysis results of PCR sequencing of cDNA after reverse transcription of Fluc and IL12, where A illustrates the agarose gel electrophoresis results of PCR amplified fragments after reverse transcription of linear and circular mRNAs, and B illustrates the sequencing results after reverse transcription of circular mRNAs expressing Fluc and IL12. The red arrow represents the specific amplification band of circular mRNA, and the sequences framed in the red box are the circularization sites of Fluc and IL12.

The experimental results showed that the specific amplification band was not present in linear mRNA group but appeared in circular mRNA group. The specific band was excised from the gel. The cDNA was recovered from the gel, and then subjected to Sanger sequencing after purification. The sequencing results showed that the PCR band was consistent with the expected band, and there was no additional base insertion or deletion near the circularization site. This demonstrated that Luciferase and IL12 have been accurately circularized.

Example 10: Analysis and Verification of Expression of Uncircularized Linear Part

This example verified that the linear RNAs produced by the Clean PIE system of the present disclosure cannot express any protein, indicating that the linear mRNAs in the present disclosure cannot produce any nonspecific translated protein before circularization.

In this example, according to the method of Example 5, the linear and circular mRNAs of FLAG-con1-SPOP in the classic PIE system as well as the linear and circular mRNAs in the Clean PIE system were transfected into 293T cells. After 24 hours, the cells were collected, lysed, and subjected to western blotting detection. Due to the presence of FLAG label after the promoter, the expressed protein can be detected by an anti-FLAG antibody.

FIGS. 25A and 25B shows the expression detection results of uncircularized linear mRNAs for preparing circular mRNAs of the classic PIE system and Clean PIE system of the present disclosure, and that of circularized circular mRNAs, where FIG. 25A shows the structures of uncircularized linear mRNAs of the classic PIE system and Clean PIE system of the present disclosure, and FIG. 25B shows the protein expression results of linear mRNAs detected by western blotting. The results showed as follows.

Under the classic PIE framework, the uncircularized mRNA can still be expressed, while under the Clean PIE framework, no expression products of uncircularized mRNAs appeared. Circularized circular RNAs from different circularization systems can be expressed, and the difference in expression level of circular mRNAs from different systems is due to different elements in the systems. This example demonstrated that the Clean PIE system of the present disclosure has a high biological safety for preparing circular mRNAs.

Example 11: Translational Regulatory Elements Enhance Expression of Circular mRNAs Through Clean PIE Circularization

In this embodiment, an insertion element, specifically, a translational regulatory element, was inserted into the Clean PIE circularization system provided in Example 3, which was ligated to the 5′ end of ev29. By adding a translational regulatory element, the expression of circular mRNAs produced by the circularization methods of the present disclosure can be enhanced. And the optimal polyAC length that is favorable to the expression of the encoded protein is obtained through screening.

The Clean PIE circularization system ligating translation regulatory element comprises structure as shown below:

-   -   T4td intron fragment II-eGFP truncated fragment II-translational         regulatory element (PolyAC)-ev29-eGFP truncated fragment I-T4td         intron fragment I.

Wherein, the nucleotide sequence of PolyAC is as shown in SEQ ID NO:1, and the circularized sequence after adding PolyAC is as shown in SEQ ID NO:2.

Circular mRNAs were prepared with the linear eGFP messenger ribonucleotide with translational regulatory expression element, according to the method of Example 3. Then, the circular mRNAs were transfected into 293T cells and the expression of eGFP was measured by flow cytometry, according to the method of Example 4.

FIG. 26 showed the expression detection results of circular mRNAs prepared by a Clean PIE system after inserting a translational regulatory element (polyAC). Result showed as follows.

As the number of inserted translational regulatory elements increases, the expression level of eGFP encoded by circular mRNAs can be significantly increased to some extent. Wherein, adding 6× polyAC achieved the greatest improvement in expression efficiency, while further increasing the length did not significantly improve the expression of the encoded protein (data for 10× polyAC).

Example 12: Regulation of the Tissue-Specific Expression of Circular mRNAs Through Clean PIE Circularization by Translational Regulatory Elements

In this example, an insertion element, specifically, a translational regulatory element for regulating specific expression of circular mRNA in organs, was ligated to the Clean PIE circularization system at the 5′ end of the IRES element. The Clean PIE system included the following structure, and each element sequence thereof can be referred to Examples 3-5:

T4td intron fragment II-LUC truncated fragment II-translational regulatory element-ev29-LUC truncated fragment I-T4td intron fragment I.

Circular mRNAs as follows were prepared according to the methods of Examples 3 to 4: EV29-LUC-3UTR (sequence set forth in SEQ ID NO: 56), EV29-LUC+1×miR-122 (sequence set forth in SEQ ID NO: 57), and EV29-LUC+3×miR-122 (sequence set forth in SEQ ID NO:58), each was encapsulated in the DLin-MC3-DMA LNP delivery system and prepared by a microfluidic device, to allow the mRNA active components in the aqueous phase to fully mix with four lipids in the organic phase to form a nano-sized circular mRNA-lipid nanoparticle complex with a high encapsulation efficiency. The specific procedure was as follows:

-   -   (1) diluting the circular mRNA stock solution to 0.4 mg/mL with         citric acid solution at pH 4.0, then weighing four lipids and         dissolving them in ethanol solution to give a total lipid         concentration of 24.4 mg/mL;     -   (2) mixing the two phases quickly using a microfluidic device,         with a total flow rate of 12 mL/min, aqueous phase (circular         mRNA)/organic phase (lipid) (v/v)=3:1;     -   (3) After the preparation completed, removing ethanol by         dialysis or tangential flow, and meanwhile replacing the         solution with PBS solution at pH 7.4, to obtain the circular         mRNA-lipid nanoparticle complex;     -   (4) detecting the particle size and polydispersity coefficient         (PDI) of the circular mRNA-lipid nanoparticle complex by dynamic         light scattering (DLS), and detecting the encapsulation         efficiency of circular mRNAs in the complex by Ribogreen.

The mice were administered by tail vein injection, and the expression of Luciferase in mice was determined after 6 hours. Specifically, mice immunized with pUC-EV29-LUC, pUC-EV29-LUC+1×miR-122, and pUC-EV29-LUC+3×miR-122 respectively were intraperitoneally injected with 0.3 mL of luciferase substrate VivoGlo luciferin (In vivo Grade, Promega), and imaged after 8 minutes to observe in vivo distribution and fluorescence intensity of expression.

FIGS. 27A and 27B shows the tissue-specific expression of circular mRNAs obtained by circularization of the Clean PIE system and regulated by the translational regulatory element, where FIG. 27A shows the expression of circular mRNAs with miR122 site injected in mice, and FIG. 27B is the frame structure of Clean PIE system with miR122 site. The experimental results showed as follows.

The luciferase in control group (EV29-luc-3UTR) mice was mainly expressed at intramuscular injection site and in liver. The circular mRNA (EV29-LUC-+1×miR-122) with a single acting site of miR-122 was mainly expressed at the tail vein injection site, and a small expression level in liver was found in individual mice, whereas the circular mRNA (EV29-LUC+3×miR-122) in which three acting sites of miR-122 were added was only expressed at the intramuscular injection site, without detection of any expression in liver. Therefore, the introduction of miR-122 binding site as a translational regulatory element in the Clean PIE system of the present disclosure can effectively avoid the expression of circular mRNAs in liver, and the more the introduced miR-122 sites, the more significant the inhibitory effects on expression in liver.

Example 13: Role of Translational Regulatory Elements in Purification of Circular mRNA

In this example, an aptamer was added to the translational regulatory element to purify the circular mRNA. Specifically, as described in the literature (Leppek K, Stoecklin G. An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins[J]. Nucleic Acids Research, 2014, 42 (2): e13-e13.), four S1m aptamer sequences were added to the translational regulatory elements of the Clean PIE system of the present disclosure, and circularization was carried out according to the method of Example 3. The structure was shown below:

T4td intron fragment II-eGFP truncated fragment II-translational regulatory element (with S1m aptamer added)-ev29-eGFP truncated fragment I-T4td intron fragment I;

S1m sequence: (SEQ ID NO: 37) AUGCGGCCGCCGACCAGAAUCAUGCAAGUGCGUAAGAUAGUCGCGGGUCGG CGGCCGCAU;

The circularized circRNA was shown in SEQ ID NO: 59.

In a LoBind tube (Eppendorf) centrifuge tube, Wash Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 2 mM vanadylribonucleosid complex RNase inhibitor (NEB), 1 tablet/10 ml Mini Complete Protease Inhibitors, EDTA-free (Roche)) was used to clean Streptavidin Sepharose High Performance (GEHealthcare) agarose gel beads. 30 μg of crude products of circular RNAs (Input) were incubated at 37° C. for 10 minutes, then rotary mixed with 100 ul agarose gel magnetic beads as well as 3 uL RNase inhibitors and incubated at 4° C. for 2-3 hours, centrifuged, unbound, and incubated continually at 4° C. for 1 hour in 50 ul of lysis buffer added with 10 mM biotin, and centrifuged to obtain the supernatant as the purified products (biotin elution).

FIG. 28 shows the detection results of gel electrophoresis of circular mRNAs purified by S1m RNA aptamer. The circular mRNAs with the aptamer can be separated from the circularization reaction system by streptomycin affinity chromatography, so that the self-splicing intron fragments and other small nucleotide fragment impurities can be removed. “Input” represented the crude products of circularized circRNA by the method of Example 7, “Biotin elution” represented the purified products, and “unbound” represented the magnetic agarose gel bead products unbond to streptomycin.

Example 14: Detection of Immunogenicity of Circularized eGFP

In this example, the expression of corresponding immune factors in A549 cells induced by circular mRNAs was detected, where the circular mRNAs were prepared by Anabaena PIE system and the Clean PIE system of the present disclosure as follows.

As provided in Example 3, circular mRNAs prepared by circularization of Anabaena PIE system and the Clean PIE system of the present disclosure were digested by RNaseR and purified by HPLC. Then, the purified circular mRNAs were transfected into A549 cells by Lipofectamine Messenger Max (Invitrogen), with the following specific operation procedure.

A549 cells were inoculated into DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution and cultured in an incubator containing 5% CO2 at 37° C. Cells were subcultured every 2 to 3 days.

(1) Cell Transfection

Before transfection, A549 cells were inoculated into a 24-well plate with 1×10⁵ cells/well, and cultured in an incubator containing 5% CO₂ at 37° C. When the cells reached 70-90% confluence, mRNAs were transfected into A549 cells by Lipofectamine MessengerMax (Invitrogen) transfection reagent at 500 ng/well, with the following specific operations.

1) Messenger MAX™ Reagent was diluted according to a dilution system shown in the following table.

TABLE 12 Reagent Volume/well MEM serum-free medium 25 μL Messenger MAX ™ Reagent 0.75 μL

After diluting and mixing, the solution was allowed to stand at room temperature for incubating for 10 min.

2) mRNAs were diluted according to a dilution system shown in the following table.

TABLE 13 Reagent Volume/well mRNA 1 μg MEM serum-free medium up to 25 μL

3) The mixed and diluted Messenger MAX™ Reagent and mRNA (1:1) was taken.

TABLE 14 Reagent Volume/well Diluted Messenger MAX ™ 25 μL Reagent Diluted mRNA 25 μL

After diluting and mixing, the solution was allowed to stand at room temperature for incubating for 5 min.

(2) 50 μL of the above mixture was taken up, and slowly add into a 24-well plate adhering to the wall, and then incubated in an incubator containing 5% CO₂ at 37° C.

(3) Lysing on the cells after 8 hours of expression was performed, and the expression level of immune response protein was verified by fluorescence quantitative PCR.

The primer sequences used by fluorescence quantitative PCR were as follows:

IFNb-F: (SEQ ID NO: 42) TGGGAGGATTCTGCATTACC IFNb-R: (SEQ ID NO: 43) CAGCATCGCTGGTTGAGA RIG-1-F: (SEQ ID NO: 44) CTCCCGGCACAGAAGTGTAT RIG-1-R: (SEQ ID NO: 45) CTTCCTCTGCCTCTGGTTTG IFNa-F: (SEQ ID NO: 46) CCATCTCTGTCCTCCATGAG IFNa-R: (SEQ ID NO: 47) ATTTCTGCTCTGACAACCTC PKR-F: (SEQ ID NO: 48) TGCAAAATGGGACAGAAAGA PKR-R: (SEQ ID NO: 49) TGATTCAGAAGCGAGTGTGC MDA5-F: (SEQ ID NO: 50) ACCAAATACAGGAGCCATGC MDA5-R: (SEQ ID NO: 51) GCGATTTCCTTCTTTTGCAG TNFa-F: (SEQ ID NO: 52) CGTCTCCTACCAGACCAAGG TNFa-R: (SEQ ID NO: 53) CCAAAGTAGACCTGCCCAGA IL-6-F: (SEQ ID NO: 54) TACCCCCAGGAGAAGATTCC IL-6-R: (SEQ ID NO: 55) GCCATCTTTGGAAGGTTCAG

The circular mRNA sequences prepared by two kinds of PIE systems were shown in the following table.

TABLE 15 SEQ ID NO: Circular mRNA sequence expressing eGFP after SEQ ID NO: 38 circularization by Clean PIE system of the present disclosure Circular mRNA sequence expressing eGFP after SEQ ID NO: 39 circularization by Anabaena PIE system

FIG. 29 shows the expression of immune factors induced by circular mRNAs prepared by using Clean PIE system (corresponding to Clean PIE in the figure) and Anabaena PIE system (corresponding to ana-PIE in the figure). The results showed as follows.

After ana-PIE underwent detestation by RNase R and purification by HPLC, although INF-0 might still lead to immune response, there was a significantly decreased immune response in the circular mRNAs prepared by the circularization system of the present disclosure compared to that by ana-PIE, proving that the circular mRNA with more accurate sequence can reduce the induction of immunogenicity.

Example 15: No Effect on In Vitro Circularization for Circular mRNAs with Deletion of Homology Arms

In this example, the effect of adding homology arms to the Clean PIE system of the present disclosure on the circularization efficiency was detected. Specifically, DGS, the amino acid unit to be optimized obtained by the method provided in Examples 1 to 2, was codon-optimized to GAT GGA TCA (ribozyme recognition site sequence) and truncated by TC (effective base pair) to form a structure in the form of T4td intron fragment II-eGFP truncated fragment II-ev29-eGFP truncated fragment I-T4td intron fragment I, while intron sequences with or without homology arms were constructed with the specific structures as follows.

Sequence information involved in this example was shown in the following table.

TABLE 16 SEQ ID NO: eGFP protein sequence: SEQ ID NO: 3 Intron fragment II (with homology SEQ ID NO: 4 arms) Intron fragment I (with homology SEQ ID NO: 5 arms) Intron fragment II (without SEQ ID NO: 6 homology arms) Intron fragment I (with homology SEQ ID NO: 7 arms) Ev29 sequence SEQ ID NO: 10

The two constructs were transcribed and circularized by the experimental method of Example 3 to obtain their circular mRNAs. The circularization results were detected by denaturing agarose gel electrophoresis. FIG. 30 shows the detection results of gel electrophoresis of circular mRNAs prepared by the Clean PIE system with or without homology arms. The results showed as follows.

Regardless of whether homology arms were included or not in the present Clean PIE system framework, circular mRNAs can realize effective circularization. This result demonstrated that homology arms can be omitted in the circularization system of the present disclosure. The reason for this is that under the frame of the present disclosure, the circularization site (ribozyme recognition site) always divided the coding gene into two parts, and the coding fragment usually did not have a very complicated secondary structure, such benign sequence separated the promoter element from the self-splicing intron sequence, thus forming a unique secondary structure, which was more conducive to the correct folding and approach of intron sequences. As a result, an effective circularization can be achieved without homology arms under the frame of the present disclosure.

Example 16: Validation of Circularization at Circularization Sites with Different Scores

This example compared the circularization efficiency of circularization sites (ribozyme recognition sites) with different scores and at different positions in the same protein sequence (IL12 human) obtained by the screening system.

Specifically, DRVF (866, score 93.8), IWS (377, score88), SGS (1021, score 88), GGS (1285, score88) and LGS (211, score 100), the amino acid units to be optimized obtained by the method provided in Examples 1 to 2, were respectively codon-optimized to the ribozyme recognition site sequences as follows: GAT CGG GTC TTT, ATT TGG TCT, TCT GGG TCT, GGT GGG TCT, and CTT GGG TCT, and truncated by TC site to form the following structure:

T4td intron fragment II-IL12 human truncated fragment II-ev29-IL 12 human truncated fragment I-T4td intron fragment I.

TABLE 17 SEQ ID NO: IL12 (human, 866 score 93.8) truncated SEQ ID NO: 27 fragment II IL12 (human, 866 score 93.8) truncated SEQ ID NO: 28 fragment I IL12 (human, 377 score 88) truncated SEQ ID NO: 29 fragment II IL12 (human, 377 score 88) truncated SEQ ID NO: 30 fragment I IL12 (human, 1021 score 88) truncated SEQ ID NO: 31 fragment II IL12 (human, 1021 score 88) truncated SEQ ID NO: 32 fragment I IL12 (human, 1285 score 88) truncated SEQ ID NO: 33 fragment II IL12 (human, 1285 score 88) truncated SEQ ID NO: 34 fragment I IL12 (human, 211 score 100) truncated SEQ ID NO: 35 fragment II IL 12 (human, 211 score 100) truncated SEQ ID NO: 36 fragment I

The two constructs were transcribed and circularized by the experimental method of Example 3 to obtain their corresponding circular mRNAs. The structure shown in FIG. 31 was obtained by denaturing agarose gel electrophoresis analysis, and the results showed as follows.

IL12 human truncated at circularization sites with different scores can be circularized, but with different circularization efficiencies. The difference of circularization efficiency may be due to different secondary structures at different open-loop positions. Therefore, it may be deduced that a small score difference of circularization site cannot properly reflect the circularization efficiency. Further evaluation is required in combination with the secondary structure in the sequence.

Example 17: Exploration of the Lowest Score Allowing for Circularization

In this embodiment, the lowest circularization score was determined by verifying circularization of truncated fragments with different scores.

Taking T4td as an example, by comparing the changed sequences at the circularization site (ribozyme recognition site) (TTGGGTCT), the circularization site sequence of eGFP sequence in Examples 6 and 7, was changed to the following base sequences with different scores for verifying circularization:

Score 100 (TTGGGTCT), score 94.2 (TCGGGTCT), score 82.4 (TAGGGTCT, ATGGGTCT), score 64.8 (AAGGGTCT, ATGGCTCT), score 47.2 (AACGGTCT, TTCATTCT), score 29.6 (AACGCTCT, AAACCGTCT, TACCCTCT).

Circularization verification was performed according to the method described in Example 7. To be specific, sequences with scores of 80 or more can be circularized with a circularization efficiency of 50% or more. In addition, the circularization test of sequences with scores of 47.2 and 64.8 showed that not all sequences can be circularized, and some sequences can be circularized but with low circularization efficiency. Further, it was difficult for the sequence with score 29.6 to circularize.

Example 18: Expression of Polypeptides Ligated in Tandem by Linker T2A

In this example, the coding regions of eGFP and firefly Luciferase were ligated in tandem by 2A peptide (T2A) encoded by a linker, and then the expression of those proteins were verified.

Specifically, according to the method of Example 3, a circular mRNA was constructed by using the Clean PIE system containing the following structure, and the circular mRNA capable of expressing eGFP and firefly Luciferase ligated in tandem by T2A was obtained.

Intron fragment II-eGFP truncated fragment II-EV29-Luciferase coding region-linker-eGFP truncated fragment I-intron fragment I, where the sequence of eGFP truncated fragment II-EV29-Luciferase coding region-linker-eGFP truncated fragment I was shown in SEQ ID NO: 40.

The obtained circular mRNAs were transfected into 293T cells by the method provided in Example 4, and the expression of eGFP and firefly Luciferase was detected by fluorescence microscope and Luciferase reporter assay kit (abcam).

FIGS. 32A through 32C show the detection results of protein expression of eGFP and firefly Luciferase expressed by circular mRNAs containing different coding regions ligated in tandem by T2A, where FIG. 32A shows the detection results of cellular immunofluorescence, FIG. 32B shows the detection results of protein expression of eGFP and firefly Luciferase, and FIG. 32C showed the structure of Clean PIE system. The results showed as follows.

As verified by the fluorescence microscope and Luciferase Reporter Assay Kit, the expression of eGFP and Luciferase was normally expressed, proving that it was feasible to link coding regions in tandem by a linker (T2A). By this method, two or more proteins can be encoded on the same circular mRNA at the same time.

Example 19: Expression of Different Target Polypeptides Ligated in Tandem by IRES

In this example, the coding regions of eGFP and firefly Luciferase were ligated in tandem by IRES, and then the expression of those proteins were verified.

According to the method of Example 3, a circular mRNA was constructed by using the Clean PIE system containing the following structure, and the circular mRNA capable of expressing eGFP and firefly Luciferase ligated in tandem by IRES was obtained.

Intron fragment II-eGFP truncated fragment II-EV29-Luciferase coding region-IRES-eGFP truncated fragment I-intron fragment I, where the sequence of eGFP truncated fragment II-EV29-Luciferase coding region-IRES-eGFP truncated fragment I was shown in SEQ ID NO: 41.

The obtained circular mRNAs were transfected into 293T cells by the method provided in Example 4, and the expression of eGFP and firefly Luciferase was detected by fluorescence microscope and Luciferase reporter assay kit (abcam).

FIGS. 33A through 33C show the detection results of protein expression of eGFP and firefly Luciferase expressed by circular mRNAs containing different coding regions ligated in tandem by IRES, where FIG. 33A shows the detection results of cellular immunofluorescence, FIG. 33B shows the detection results of protein expression of eGFP and firefly Luciferase, and FIG. 33C shows the structure of Clean PIE system. The results showed as follows.

eGFP and Luciferase were ligated in tandem in the same circular mRNA by different IRES, enabling an effective circularization. As verified by the fluorescent microscope and Luciferase Reporter Assay Kit, GFP and Luciferase were normally expressed, proving that it was feasible to express different coding regions ligated in tandem by IRES. By this method, two or more proteins can be encoded on the same circular mRNA at the same time.

The above examples of the present disclosure are only for clearly illustrating the present disclosure, and are not limitations on the embodiments of the present disclosure. For those of ordinary skill in the art to which the present disclosure belongs, other changes or variations can be made on the basis of the above description. It is not necessary and impossible to exhaust all the embodiments herein. Any modification, equivalent substitution, improvement, and the like made within the spirit and principle of the present disclosure should be included within the scope of the claims of the present disclosure. 

1. A recombinant nucleic acid molecule for preparing a circular RNA in vitro, the recombinant nucleic acid molecule being selected from any one of (i) to (ii): (i) the recombinant nucleic acid molecule comprising elements arranged in the following order in the 5′ to 3′ direction: an intron fragment II, a truncated fragment II of a coding element, a translation initiation element, a truncated fragment I of a coding element, and an intron fragment I, wherein the truncated fragment II is directly joined with intron fragment II without any intervening sequences, and truncated fragment I is directly joined with intron fragment I without any intervening sequences, wherein a 3′ end of the truncated fragment I of the coding element comprises a ribozyme recognition site I that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment I of the coding element, a 5′ end of the truncated fragment II of the coding element comprises a ribozyme recognition site II that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment II of the coding element, wherein the nucleotide sequence of the truncated fragment I of the coding element and the nucleotide sequence of the truncated fragment II of the coding element form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction; the nucleotide sequence of the truncated fragment I of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence; and the nucleotide sequence of the truncated fragment II of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence, wherein the nucleotide sequence of the intron fragment I and the nucleotide sequence of the intron fragment II form an intron sequence in the 5′ to 3′ direction; the nucleotide sequence of the intron fragment I comprises a partial sequence close to the 5′ direction in the intron sequence; and the nucleotide sequence of the intron fragment II comprises a remaining partial sequence close to the 3′ direction in the intron sequence; or (ii) the recombinant nucleic acid molecule comprising elements arranged in the following order in the 5′ to 3′ direction: an intron fragment III, a truncated fragment IV of a coding element, a translation initiation element, a truncated fragment III of a coding element, and an intron fragment IV, wherein the truncated fragment IV is directly joined with intron fragment III without any intervening sequences, and truncated fragment III is directly joined with intron fragment IV without any intervening sequences, wherein a 3′ end of the truncated fragment III of the coding element comprises a ribozyme recognition site IV that consists of a second predetermined number of nucleotides located at the 3′ end of the truncated fragment III of coding element, a 5′ end of the truncated fragment IV of the coding element comprises a ribozyme recognition site III that consists of a first predetermined number of nucleotides located at the 5′ end of the truncated fragment IV of coding element, wherein the nucleotide sequence of the truncated fragment III of the coding element and the nucleotide sequence of the truncated fragment IV of the coding element form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction; the nucleotide sequence of the truncated fragment III of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence; and the nucleotide sequence of the truncated fragment IV of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence, wherein the sequence of the intron fragment III is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment I, and the sequence of intron fragment IV is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment II; the sequence of the ribozyme recognition site III is a reverse sequence of the nucleotide sequence of the ribozyme recognition site I, and the sequence of the ribozyme recognition site IV is a reverse sequence of the nucleotide sequence of the ribozyme recognition site II.
 2. The recombinant nucleic acid molecule of claim 1, wherein the intron fragment I and the intron fragment II are derived from Group I intron, the ribozyme recognition site I is derived from a natural exon sequence ligated to the 5′ end of the intron fragment I, and the ribozyme recognition site II is derived from a natural exon sequence ligated to the 3′ end of the intron fragment II; and optionally, the Group I intron is derived from any one of the following Group I introns: phage T4 td gene, Anabaena tRNA^(Leu), TpaCOX2, and Ptu.
 3. The recombinant nucleic acid molecule of claim 1, wherein the first predetermined number of nucleotides is selected from 3 to 100 nucleotides, preferably 3 to 50 nucleotides, and more preferably 3 to 10 nucleotides; or wherein the second predetermined number of nucleotides is selected from 1 to 100 nucleotides, preferably 1 to 50 nucleotides, and more preferably 1 to 10 nucleotides; or wherein a sum of the first predetermined number and the second predetermined number is not equal to 3y, wherein y≥1 and y is an integer.
 4. The recombinant nucleic acid molecule of claim 1, wherein the translation initiation element comprises a sequence with an activity of initiating translation of an editing region; and optionally, the sequence with the activity of initiating translation of an editing region is selected from one of or a combination of two or more of the following: an IRES sequence, a 5′ UTR sequence, a Kozak sequence, a sequence with m⁶A modification, and a complementary sequence of ribosome 18S rRNA.
 5. The recombinant nucleic acid molecule of claim 1, wherein the recombinant nucleic acid molecule is used for preparing a circular RNA comprising a coding element, wherein the coding element in the circular RNA comprises a coding region 1, optionally (a) at least one coding region 2, and optionally (b) at least one coding region 3; the truncated fragment I of the coding element and the truncated fragment II of the coding element form the coding region 1, optionally (a) the at least one coding region 2, and optionally (b) the at least one coding region 3; the recombinant nucleic acid molecule comprises elements arranged in the order shown in any one of (i) to (iv) in the 5′ to 3′ direction: (i) the intron fragment II, the truncated fragment II of the coding region 1, the at least one coding region 2, the translation initiation element, the truncated fragment I of the coding region 1, and the intron fragment II; (ii) the intron fragment II, the truncated fragment II of the coding region 1, the translation initiation element, the at least one coding region 3, the truncated fragment I of coding region 1, and the intron fragment I; (iii) the intron fragment II, the truncated fragment II of the coding region 1, the at least one coding region 2, the translation initiation element, the at least one coding region 3, the truncated fragment I of the coding region 1, and the intron fragment I; (iv) the intron fragment II, the truncated fragment II of the coding region 1, the translation initiation element, the truncated fragment I of the coding region 1, and the intron fragment I; alternatively, the truncated fragment III of the coding element and the truncated fragment IV of the coding element form the coding region 1, optionally (a) the at least one coding region 2, and optionally (b) the at least one coding region 3; the recombinant nucleic acid molecule comprises elements arranged in the order shown in any one of (v) to (viii) in the 5′ to 3′ direction: (v) the intron fragment III, the truncated fragment IV of the coding region 1, the at least one coding region 2, the translation initiation element, the truncated fragment III of the coding region 1, and the intron fragment IV; (vi) the intron fragment III, the truncated fragment IV of the coding region 1, the translation initiation element, the at least one coding region 3, the truncated fragment III of the coding region 1, and the intron fragment IV; (vii) the intron fragment III, the truncated fragment IV of the coding region 1, the at least one coding region 2, the translation initiation element, the at least one coding region 3, the truncated fragment III of the coding region 1, and the intron fragment IV; (viii) the intron fragment III, the truncated fragment IV of the coding region 1, the translation initiation element, the truncated fragment III of the coding region 1, and the intron fragment IV; wherein the coding region 1, each coding region 2 and each coding region 3 encode any type of target polypeptides independently of one another; or wherein the recombinant nucleic acid molecule comprises one or both elements of (i) to (ii): (i) a linker located between the truncated fragment II of the coding region 1 and the coding region 2; (ii) a linker located between the coding region 3 and the truncated fragment I of the coding region 1; alternatively, the recombinant nucleic acid molecule comprises one or both elements of (iii) to (iv): (iii) a linker located between the truncated fragment IV of the coding region 1 and the coding region 2; (iv) a linker located between the coding region 3 and the truncated fragment III of the coding region 1; optionally, the coding region 2 has a quantity of at least 2, and the recombinant nucleic acid molecule comprises a linker located between any two coding regions 2; optionally, the coding region 3 has a quantity of at least 2, and the recombinant nucleic acid molecule comprises a linker located between any two coding regions 3; and preferably, the linker is a polynucleotide encoding a 2A peptide.
 6. The recombinant nucleic acid molecule of claim 1, wherein the recombinant nucleic acid molecule is used for preparing a circular RNA comprising a coding element; wherein the coding element in the circular RNA comprises a coding region 1, at least one coding region 4, and a translation initiation element located between any two adjacent coding regions; the truncated fragment I of the coding element and the truncated fragment II of the coding element form the coding region 1, the at least one coding region 4, and the translation initiation element located between any two adjacent coding regions; or, the truncated fragment III of the coding element and the truncated fragment IV of the coding element form the coding region 1, the at least one coding region 4, and the translation initiation element located between any two adjacent coding regions.
 7. The recombinant nucleic acid molecule of claim 1, wherein the target polypeptide is a human-derived protein or non-human derived protein; and optionally, the target polypeptide is selected from one of or a combination of two or more of the following: an antigen, an antibody, an antigen-binding fragment, a fluorescent protein, a protein with therapeutic activity, and a protein with gene-editing activity; or wherein the recombinant nucleic acid molecule further comprises an insertion element between the truncated fragment II and the translation initiation element, or an insertion element between the truncated fragment IV and the translation initiation element; the insertion element is at least one selected from the group consisting of (i) to (iii): (i) a transcriptional regulatory element, (ii) a translational regulatory element, and (iii) a purification element; preferably, the insertion element is ligated to the 5′ end of any translation initiation element; optionally, the insertion element comprises a sequence selected from one of or a combination of two or more of the following: an untranslated region sequence, a polyA sequence, an aptamer sequence, a riboswitch sequence, and a transcriptional regulator-binding sequence; or wherein the recombinant nucleic acid molecule further comprises a 5′ homologous arm and a 3′ homologous arm, and the nucleotide sequence of the 5′ homologous arm is complementary pairing with the nucleotide sequence of the 3′ homologous arm; the 5′ homologous arm is ligated to the 5′ end of the intron fragment II, and the 3′ homologous arm is ligated to the 3′ end of the intron fragment I; or, the 5′ homologous arm is ligated to the 5′ end of the intron fragment III, and the 3′ homologous arm is ligated to the 3′ end of the 3′ intron fragment IV; or wherein a nucleotide sequence derived from an exon is not comprised in any one of the intron fragment I, the translation initiation element, or the intron fragment II, or between any two of the intron fragment I, the truncated fragment II of the coding element, the translation initiation element, the truncated fragment I of the coding element, and the intron fragment II; or, a reverse sequence or reverse complementary sequence of a nucleotide sequence derived from an exon is not comprised in any one of the intron fragment III, the translation initiation element, and the intron fragment IV, or between any two of the intron fragment III, the truncated fragment IV of the coding element, the translation initiation element, the truncated fragment III of the coding element, and the intron fragment IV.
 8. A recombinant expression vector, comprising the recombinant nucleic acid molecule of claim
 1. 9. A method for preparing a circular RNA in vitro, comprising the steps of: transcribing the recombinant nucleic acid molecule of claim 1 to form a cyclization precursor nucleic acid molecule; cyclizing the cyclization precursor nucleic acid to obtain the circular RNA; and optionally, further comprising the step of purifying the circular RNA.
 10. A circular RNA, comprises elements arranged in the following order in the 5′ to 3′ direction: a translation initiation element, a coding element for encoding at least one target polypeptide; optionally, the circular RNA comprises an insertion element located between the 5′ end of the translation initiation element and the 3′ end of the coding element; optionally, the translation initiation element comprises a sequence with an activity of initiating translation of an editing region, the target polypeptide is a human-derived protein or non-human derived protein, and the insertion element is at least one selected from the group consisting of (i) to (iii): (i) a transcriptional regulatory element, (ii) a translational regulatory element, and (iii) a purification element.
 11. The circular RNA of claim 10, wherein the coding element of the circular RNA comprises a coding region 1, and at least one in the group consisting of (i) to (ii): (i) at least one coding region 2, and (ii) at least one coding region 3; each coding region encodes any type of target polypeptide independently of one another; preferably, any two adjacent coding regions are linked by a linker; or wherein the coding element of the circular RNA comprises the coding region 1 and at least one coding region 4, and the 5′ end of any coding region is ligated to a translation initiation element; or wherein the insertion element is ligated to the 5′ end of any translation initiation element.
 12. A composition comprising the circular RNA of claim 10; optionally, the composition further comprises one or more pharmaceutically acceptable carriers; and optionally, the pharmaceutically acceptable carrier is selected from a lipid, a polymer or a lipid-polymer complex.
 13. A method for expressing a target polypeptide in a cell, wherein the method comprises the step of delivering the circular RNA of claim 10 into the cell.
 14. A method for preventing or treating diseases, wherein the method comprises administering the circular RNA of claim 10 to a subject. 15.-20. (canceled)
 21. The recombinant nucleic acid molecule of claim 1, wherein during circularization, the truncated fragment I is directly joined with the truncated fragment II, without any intervening sequences.
 22. The recombinant nucleic acid molecule of claim 1, wherein during circularization, the truncated fragment III is directly joined with the truncated fragment IV, without any intervening sequences.
 23. The recombinant nucleic acid molecule of claim 1, wherein the recombinant nucleic acid molecule further comprises an IRES sequence that is flanked by truncated fragment I and truncated fragment II.
 24. The recombinant nucleic acid molecule of claim 1, wherein after circularization, the ligation junction is located within the coding element sequence. 